U.S. patent number 8,414,892 [Application Number 12/721,798] was granted by the patent office on 2013-04-09 for uses of monoclonal antibody 8h9.
This patent grant is currently assigned to Sloan-Kettering Institute for Cancer Research. The grantee listed for this patent is Nai-Kong V. Cheung. Invention is credited to Nai-Kong V. Cheung.
United States Patent |
8,414,892 |
Cheung |
April 9, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Uses of monoclonal antibody 8H9
Abstract
This invention provides a composition comprising an effective
amount of monoclonal antibody 8H9 or a derivative thereof and a
suitable carrier. This invention provides a pharmaceutical
composition comprising an effective amount of monoclonal antibody
8H9 or a derivative thereof and a pharmaceutically acceptable
carrier. This invention also provides an antibody other than the
monoclonal antibody 8H9 comprising the complementary determining
regions of monoclonal antibody 8H9 or a derivative thereof, capable
of binding to the same antigen as the monoclonal antibody 8H9. This
invention provides a substance capable of competitively inhibiting
the binding of monoclonal antibody 8H9. This invention also
provides an isolated scFv of monoclonal antibody 8H9 or a
derivative thereof. This invention also provides the 8H9 antigen.
This invention also provides different uses of the monoclonal
antibody 8H9 or its derivative.
Inventors: |
Cheung; Nai-Kong V. (New York,
NY) |
Applicant: |
Name |
City |
State |
Country |
Type |
Cheung; Nai-Kong V. |
New York |
NY |
US |
|
|
Assignee: |
Sloan-Kettering Institute for
Cancer Research (New York, NY)
|
Family
ID: |
43062436 |
Appl.
No.: |
12/721,798 |
Filed: |
March 11, 2010 |
Prior Publication Data
|
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|
|
Document
Identifier |
Publication Date |
|
US 20100284920 A1 |
Nov 11, 2010 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10505658 |
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7740845 |
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PCT/US03/07004 |
Mar 6, 2003 |
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10273762 |
Oct 17, 2002 |
7666424 |
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10097558 |
Mar 8, 2002 |
7737258 |
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PCT/US02/33331 |
Oct 17, 2002 |
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10097558 |
Mar 8, 2002 |
7737258 |
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PCT/US01/32565 |
Oct 18, 2001 |
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60241344 |
Oct 18, 2000 |
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60330396 |
Oct 17, 2001 |
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Current U.S.
Class: |
424/141.1 |
Current CPC
Class: |
C07K
16/4208 (20130101); C07K 16/4266 (20130101); C07K
16/3053 (20130101); C07K 16/28 (20130101); A61K
47/6865 (20170801); A61K 49/0006 (20130101); C07K
16/30 (20130101); C07K 16/18 (20130101); A61K
47/6873 (20170801); A61K 49/0004 (20130101); C07K
16/00 (20130101); C07K 2317/24 (20130101); C07K
2319/00 (20130101); C07K 2317/622 (20130101); C07K
2317/76 (20130101); A01K 2217/05 (20130101); A61K
2039/505 (20130101); C07K 2317/732 (20130101); C07K
2317/34 (20130101) |
Current International
Class: |
A61K
39/395 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 03/075846 |
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Sep 2003 |
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WO |
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WO 2004/050849 |
|
Jun 2004 |
|
WO |
|
Other References
Modak et al (Proceedings of ASCO, 1998, 17:445a). cited by examiner
.
Savage et al (Br J Cancer, 1993, 68: 738-742). cited by examiner
.
Cheung, N. K. V. et al., Anti-GD2 antibody treatment of minimal
residual stage 4 neuroblastoma diagnosed at more than 1 year of
age. J. Clin. Oncol., 16:3053-3060, 1998. (Exhibit 1). cited by
applicant .
Yu, A. et al. et al., Phase I trial of a human-mouse chimeric
anti-disialoganglioside monoclonal antibody ch14.18 in patients
with refractory neuroblastoma and osteosarcoma. J. Clin. Oncol.,
16:2169-2180, 1998. (Exhibit 2). cited by applicant .
Jurcic, J. G. et al., Sequential targeted therapy for acute
promyelocytic leukemia with all-trans retinoic acid and anti-CD33
monoclonal antibody M195. Leuk., 9:244-248, 1995. (Exhibit 3).
cited by applicant .
Czuczman, M. S. et al. Treatment of patients with low-grade B-cell
lymphoma with the combination of chimeric anti-CD20 monoclonal
antibody and CHOP chemotherapy. J. Clin. Oncol., 17:268-276, 1999.
(Exhibit 4). cited by applicant .
Garin-Chesa, P. et al, Immunohistochemical analysis of neural cell
adhesion molecules. Differential expressionin small round cell
tumors of childhood and adolescence. Am. J. Pathol., 139:275-286,
1991. (Exhibit 5). cited by applicant .
Ritter, G. et al, Ganglioside antigens expressed by human cancer
cells. Semin. Cancer. Biol., 2:401-409, 1991. (Exhibit 6). cited by
applicant .
Ylagan et al., CD44 expression in astrocytic tumors. Modern
Pathology, 10:1239-1246, 1997. (Exhibit 7). cited by applicant
.
Kuan, C. T. et al., 125I-labeled anti-epidermal growth factor
receptor vIII single-chain Fv exhibits specific and high-level
targeting of glioma xenografts. Clin. Can. Res., 5:1539-1549, 1999.
(Exhibit 8). cited by applicant .
Richardson, R. B. et al., Radioimmunolocalization of human brain
tumors. Biodistribution of radiolabelled monoclonal antibody UJ13A.
Eur J Nucl Med, 12:313-320, 1986. (Exhibit 9). cited by applicant
.
Papanastassiou, V. et al., Treatment of recurrent and cystic
malignant gliomas by a single intracavitary injection of
131I-monoclonal antibody: Feasibility, pharmacokinetics and
dosimetry. Br. J. Cancer, 67:144-151, 1993. (Exhibit 10). cited by
applicant .
Celis, E. et al., Induction of anti-tumor cytotoxic T lymphocytes
in normal humans using primary cultures and synthetic peptide
epitopes. Proc. Natl. Acad. Sci. USA, 91:2105-2109, 1994. (Exhibit
11). cited by applicant .
Riva, P. et al., 131I radioconjugated antibodies for the
locoregional radioimmunotherapy of high-grade malignant glioma-
phase I and II study. Acta Oncol, 38:351-359, 1999. (Exhibit 12).
cited by applicant .
Heiner, J. et al., Localization of GD2 specific monoclonal antibody
in human osteogenic sarcoma. Cancer Res., 47:5377-5381, 1987.
(Exhibit 13). cited by applicant .
Spendlove, I. et al., Decay accelerating factor (CD55): a target
for cancer vaccines! Cancer Res., 59:2282-2286, 1999. (Exhibit 14).
cited by applicant .
Weidner, N. et al., Immunohistochemical profile of monoclonal
antibody O13 that recognizes glycoprotein 930/32MIC2 and is useful
in diagnosing ewing's sarcoma and peripheral neuroepithelioma.
American Journal of Surgical Pathology, 18:486-494, 1994. (Exhibit
15). cited by applicant .
Hatzubai, A. et al., The use of a monoclonal anti-idiotype antibody
to study the biology of human B-cell lymphoma. J. Immunol.,
126:2397-2402, 1981. (Exhibit 16). cited by applicant .
Cheung, N. K. et al., Monoclonal antibodies to a glycolipid antigen
on human neuroblastoma cells. Cancer Res., 45:2642-2649, 1985.
(Exhibit 17). cited by applicant .
Kramer, K. et al., Prognostic value of TrkA protein detection my
monoclonal antibody 5C3 in Neuroblastoma. Clin. Can. Res.,
2:1361-1367, 1996. (Exhibit 18). cited by applicant .
Hecht, T. T. et al., Production and characterization of a
monoclonal antibody that binds reed-sternberg cells. J. Immunol.,
134:4231-4236, 1985. (Exhibit 19). cited by applicant .
Seeger, R. C. et al., Danon, Y. L., Rayner, S. A., Hoover, F.
Definition of a Thy-1 deteminant on human neuroblastoma, glioma,
sarcoma, and teratoma cells with a monoclonal antibody. J.
Immunol., 128:983-989, 1982. (Exhibit 20). cited by applicant .
Kaaijk, P. et al., Expression of CD44 splice variants in human
primary brain tumors. Journal of Neuro-Oncology, 26:185-190, 1995.
(Exhibit 21). cited by applicant .
Wikstrand, C. J. et al., Lactotetraose series ganglioside
3',6'-isoLD1 in tumors of central nervous and other systems in
vitro and in vivo. Cancer Res., 53:120-126, 1993. (Exhibit 22).
cited by applicant .
Pappo, A. et al., biology and treatment. Pediatr. Clin. North Am.,
44:953-972, 1997. (Exhibit 23). cited by applicant .
Fujisawa, T. et al., A monoclonal antibody with selective
immunoreactivity for neuroblastoma and rhabdomyosarcoma. Proc. Am.
Assoc. Cancer Res., 30:345, 1989. (Exhibit 24). cited by applicant
.
Wikstrand, C. J. et al., Monoclonal Antibodies against EGFRvIII are
Tumor Specific and React with Breast and Lung Carcinomas and
Malignant Gliomas. Cancer Res., 55:3140-48, 1995. (Exhibit 25).
cited by applicant .
Kishima, H. et al., Monoclonal antibody ONS-21 recognizes integrin
a3 in gliomas and gliomas and medulloblastomas. Br. J. Cancer,
79:333-339, 1998. (Exhibit 26). cited by applicant .
Moriuchi, S. et al., Characterization of a new mouse monoclonal
antibody (ONS-M21) reactive with both medulloblastomas and gliomas.
Br. J. Cancer, 68:831-837, 1993. (Exhibit 27). cited by applicant
.
Kondo, S. et al., Human glioma-specific antigens detected by
monoclonal antibodies. Neurosurgery, 30:506-511, 1992. (Exhibit
28). cited by applicant .
Dastidar, S. G. et al., Monoclonal antibody against human
glioblastoma multiforme (U-087Mg) immunoprecipitates a protein of
monoclonal mass 38Kda and inhibits tumor growth in nude mice. J
Neuroimmuno, 56:91-98, 1995. (Exhibit 29). cited by applicant .
Mihara, Y. et al., Monoclonal antibody against ependymoma-derived
cell line. Journal of Neuro-Oncology, 12:1-11, 1992. (Exhibit 30).
cited by applicant .
Daghighian, F. et al., Development of a method to measure kinetics
of radiolabeled monoclonal antibody in human tumour with
applications to microdosimetry: positron emission tomography
studies of iodine-124 labeled 3F8 monoclonal antibody in glioma.
Eur J Nucl Med, 20:402-409, 1993. (Exhibit 31). cited by applicant
.
Plate, K. H. et al., Platelet derived growth factor b is induced
during tumor development and upregulated during tumor progressing
in endothelial cells in human gliomas. Lab. Invest., 67:529-534,
1992. (Exhibit 32). cited by applicant .
Yang, H. S. et al., Expression of 300-kilodalton intermediate
filament-associated protein distinguishes human glioma cells from
normal astrocytes. Proceedings of the National Academy of Sciences
of the United States of America, 90:8534-8537, 1993. (Exhibit 33).
cited by applicant .
Koehler G et al., Continuous culture of fused cells secreting
antibody of pre-defined specificity. Nature 256:495-496, 1975
(Exhibit 34). cited by applicant .
Moffat R et al., Clinical utility of external immunoscintigraphy
with the IMMU-4 technetium-99m Fab' antibody fragment in patients
undergoing surgery for carcinoma of the colon and rectum:results of
a pivotal, phase III trial. The Immunomedics Study Group. J Clin
Oncol 14(8):2295-2305, 1996 (Exhibit 35). cited by applicant .
Maloney DG et al., IDEC-C2B8: Results of a phase I multiple-dose
trial in patients with relapsed non-hodgkin's lymphoma. J Clin
Oncol 15:3266-3274, 1997 (Exhibit 36). cited by applicant .
Cobleigh MA et al., Multinational study of the efficacy and safety
of humanized anti-HER2 monoclonal antibody in woman who have
HER2-overexpressing metastatic breast cancer that has progressed
after chemotherapy for metastatic disease. J Clin Oncol
17:2639-2648, 1999 (Exhibit 37). cited by applicant .
Meredith RF et al., Phase II study of dual 131I-labeled monoclonal
antibody therapy with interferon in patients with metastatic
colorectal cancer. Clin Can Res 2:1811-1818, 1996 (Exhibit 38).
cited by applicant .
Yeh SD et al., Radioimmunodetection of neuroblastoma with
iodine-131-3F8: Correlation with biopsy,
iodine-131-Metaiodobenzylguanidine (MIBG) and standard diagnostic
modalities. J Nucl Med 32:769-776, 1991 (Exhibit 39). cited by
applicant .
Wheldon TE et al., The curability of tumors of differing size by
targeted radiotherapy using 131-I or 90-Y. Radiother Oncol
21:91-99, 1991 (Exhibit 40). cited by applicant .
Wilder RB et al., Radioimmunotherapy: recent results and future
directions. J Clin Oncol 14:1383-1400, 1996 (Exhibit 41). cited by
applicant .
Zalutsky MR et al., Radioimmunotherapy of neoplastic meningitis in
rats using an alpha-particle-emitting immunoconjugate. Cancer Res
54:4719-4725, 1994 (Exhibit 42). cited by applicant .
McDevitt MR et al., Radioimmunotherapy with alpha-emitting
nuclides. Eur J Nucl Med 25:1341-1351, 1998 (Exhibit 43). cited by
applicant .
DeNardo SJ et al., Antibody phage libraries for the next generation
of tumor targeting radioimmunotherapeutics. Clin Can Res
5:3213s-3218s, 1999 (Exhibit 44). cited by applicant .
DeNardo SJ et al., Phage Library-derived human anti-TETA anti
anti-DOTA ScFv for pretargeting RIT. Hybridoma 18:13-21, 1999
(Exhibit 45). cited by applicant .
Eshhar Z et al., Specific activation and targeting of cytotoxic
lymphocytes through chimeric single chains consisting of
antibody-binding domains and the or zeta subunits of the
immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA
90:720-24, 1993 (Exhibit 46). cited by applicant .
Altenschmidt U et al., Cytolysis of tumor cells expressing the
Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted I
T lymphocytes. Clin Can Res 2:1001-1008, 1996 (Exhibit 47). cited
by applicant .
Krause A et al., Antigen-dependent CD-28 signaling enhances
survival and proliferation in genetically modified activated human
primary T lymphocytes. J Exp Med 188:619-626, 1998 (Exhibit 48).
cited by applicant .
Price MR et al., Characteristics of the cell surface antigen p72,
associated with a variety of human tumors and mitogen-stimulated
T-lymnphoblasts. FEBS Letters 171:31-35, 1984 (Exhibit 49). cited
by applicant .
Gorlick R et al., Expression of HER2/erbB-2 correlates with
survival in osteosarcoma. J Clin Oncol 17:2781-2788, 1999 (Exhibit
50). cited by applicant .
Cheung NK et al., Detection of metastatic neuroblastoma in bone
marrow: when is routine marrow histology insensitive? J Clin Oncol
15:2807-2817, 1997 (Exhibit 51). cited by applicant .
Ghossein RA et al., Detection of circulating prostatic tumor cells
using immunobead reverse transcriptase polymerase chain reaction
for prostatic specific membrane antigen mRNA. Diag Mol Path
8:59-65, 1999 (Exhibit 52). cited by applicant .
Leung W et al., Frequent detection of tumor cells in hematopoietic
grafts in neuroblastoma and ewing's sarcoma. Bone Marrow Transpl
22:971-979, 1998 (Exhibit 53). cited by applicant .
Mueller BM et al., Enhancement of antibody-dependent cytotoxicity
with a chimeric anti-GD2 antibody. J Immunol 144:1382-1386, 1990
(Exhibit 54). cited by applicant .
Santos AD et al., Generation and characterization of a single
gene-encoded single-chain-tetravalent antitumor antibody. Clin Can
Res 5:3118s-3123s, 1999 (Exhibit 55). cited by applicant .
Guo HF et al., Recombinant anti-ganglioside GD2 scFv-streptavidin
fusion protein for tumor pretargeting. Proc Am Assoc Cancer Res
37:469, 1996 (abstract) (Exhibit 56). cited by applicant .
Fagnou C et al., Presence of tumor cells in bone marrow but not in
blood is associated with adverse prognosis in patients with ewing's
tumor. J Clin Oncol 16:1707-1711, 1998 (Exhibit 57). cited by
applicant .
Munn DH et al., Interleukin-2 enhancement of monoclonal
antibody-mediated cellular cytotoxicity (ADCC) against human
melanoma. Cancer Res 47:6600-6605, 1987 (Exhibit 58). cited by
applicant .
Hank JA et al., Augmentation of antibody dependent cell mediated
cytotoxicity following in vivo therapy with recombinant
interleukin-2. Cancer Res 50:5234-5239, 1990 (Exhibit 59). cited by
applicant .
Kushner BH et al., GM-CSF enhances 3F8 monoclonal
antibody-dependent cellular cytotoxicity against human melanoma and
neuroblastoma. Blood 73:1936-1941, 1989 (Exhibit 60). cited by
applicant .
Saarinen UM et al., Eradication of neuroblastoma cells in vitro by
monoclonal antibody and human complement: method for purging
autologous bone marrow. Cancer Res 45:5969-5975, 1985 (Exhibit 62).
cited by applicant .
Munn DH et al., Antibody-dependent antitumor cytotoxicity by human
monocytes cultured with recombinant macrophage colony-stimulating
factor. Induction of efficient antibody-mediated antitumor
cytotoxicity not detected by isotope release assays. J Exp Med
170:511-526, 1989 (Exhibit 63). cited by applicant .
Munn DH et al., Phagocytosis of tumor cells by human monocytes
cultured in recombinant macrophage colony-stimulating factor. J Exp
Med 172:231-237, 1990 (Exhibit 64). cited by applicant .
Sabzevari H et al., A recombinant antibody-interleukin 2 fusion
protein suppresses growth of hepatic human neuroblastoma metastases
in severe combined immunodeficiency mice. Proceeds of the National
Academy of Science USA 91:9626-9630, 1994 (Exhibit 65). cited by
applicant .
Mujoo K et al., A potent and specific immunotoxin for tumor cells
expressing disialoganglioside GD2. Cancer Immunol Immunother
34:198-204, 1991 (Exhibit 66). cited by applicant .
Gottstein C et al., Antidisialoganglioside Ricin A-chain
immunotoxins show potent anti-tumor effects in vitro and in a
disseminated human neuroblastoma severe combined immunodeficiency
mouse model. Cancer Res 54:6186-6193, 1994 (Exhibit 67). cited by
applicant .
Holzer U et al.,
Superantigen-staphylococcal-enterotoxin-A-dependent and
antibody-targeted lysis of GD2-positive neruoblastoma cells. Cancer
Immunol Immunother 41:129-136, 1995 (Exhibit 68). cited by
applicant .
Cheung NK et al., Ganglioside GD2 specific monoclonal antibody
3F8--a phase I study in patients with neuroblastoma and malignant
melanoma. J Clin Oncol 5:1430-1440, 1987 (Exhibit 69). cited by
applicant .
Cheung NK et al., Reassessment of patient response to monoclonal
antibody 3F8. J Clin Oncol 10:671-672, 1992 (Exhibit 70). cited by
applicant .
Murray JL et al., Phase I trial of murine monoclonal antibody 14G2a
administered by prolonged intravenous infusion in patients with
neuroectodermal tumors. J Clin Oncol 12:184-193, 1994 (Exhibit 71).
cited by applicant .
Uttenreuther-Fischer MM et al., Pharmacokinetics of
anti-ganglioside GD2 mAb 14G2a in phase 1 trial in pediatric cancer
patients. Cancer Immunol Immunother 41:29-36, 1995 (Exhibit 72).
cited by applicant .
Handgretinger R et al., A phase I study of neuroblastoma with the
anti-ganglioside GD2 antibody 14.G2a. Cancer Immunol Immunother
35:199-204, 1992 (Exhibit 73). cited by applicant .
Miraldi FD et al., Diagnostic imaging of human neuroblastoma with
radiolabeled antibody. Radiology 161:413-418, 1986 (Exhibit 74).
cited by applicant .
Arbit E et al., Quantitative Immunoimaging of gliomas in humans
with anti-ganglioside monoclonal antibodies. J Neurosurg 76:399a,
1991 (Exhibit 75). cited by applicant .
Grant SC et al., Radioimmunodetection of small-cell lung cancer
using the anti-GD2 ganglioside monoclonal antibody 3F8: a pilot
trial. Eur J Nucl Med 23:145-149, 1996 (Exhibit 76). cited by
applicant .
Larson SM et al., PET scanning of iodine-124-3F8 as an approach to
tumor dosimetry during treatment planning for radioimmunotherapy in
a child with neuroblastoma. J Nucl Med 33:2020-2023, 1992 (Exhibit
77). cited by applicant .
Pentlow KS et al., Quantitative imaging of I-124 using positron
emission tomography with applications to radioimmunodiagnosis and
radioimmunotherapy. Medical Physics 18:357-366, 1991 (Exhibit 78).
cited by applicant .
Pentlow KS et al., Quantitative imaging of iodine-124 with PET. J
Nucl Med 37:1557-1562, 1996 (Exhibit 79). cited by applicant .
Saleh MN et al., A phase I trial of the murine monoclonal anti-GD2
antibody 14.G2a in metastatic melanoma. Cancer Res 52:4342-4347,
1992 (Exhibit 80). cited by applicant .
Cheung NK et al., Antibody response to murine anti-GD2 monoclonal
antibodies: Correlation with patient survival. Cancer Res
54:2228-2233, 1994 (Exhibit 81). cited by applicant .
Drengler RL et al., Phase I and pharmacokinetic trial of oral
irinotecan administered daily for 5 days every 3 weeks in patients
with solid tumors. J Clin Oncol 17:685-696, 1999 (Exhibit 82).
cited by applicant .
Cheung NK et al., Complete tumor ablation with iodine
131-radiolabeled disialoganglioside GD2 specific monoclonal
antibody against human neuroblastoma xenografted in nude mice. J
Natl Cancer Inst 77:739-745, 1986 (Exhibit 83). cited by applicant
.
Cheung NK et al., Disialoganglioside GD2 anti-idiotypic monoclonal
antibodies. Int J Cancer 54:499-505, 1993 (Exhibit 84). cited by
applicant .
Loh A et al., A pharmacokinetic model of 131I-G250 antibody in
patients with renal cell carcinoma. J Nucl Med 3:484-489, 1998
(Exhibit 85). cited by applicant .
Kolbert KS et al., Implementation and evaluation of
patient-specific three dimensional internal dosimetry. J Nucl Med
38:301-308, 1997 (Exhibit 86). cited by applicant .
Sgouros G et al., Bone marrow dosimetry: Regional variability of
marrow-localizing antibody. J Nucl Med 37:695-698, 1996 (Exhibit
87). cited by applicant .
Sgouros G et al., Marrow and whole-body absorbed dose vs marrow
toxicity following 131I-G250 antibody therapy in patients with
renal-cell carcinoma. J Nucl Med 38:252P, 1997 (Exhibit 88). cited
by applicant .
Furhang EE et al., Radionuclide photon dose kernels for internal
emitter dosimetry. Medical Physics 23:759-764, 1996 (Exhibit 89).
cited by applicant .
Furhang EE et al., A monte carlo approach to patient-specific
dosimetry. Medical Physics 23:1523-1529, 1996 (Exhibit 90). cited
by applicant .
Furhang EE et al., Implementation of a monte carlo dosimetry method
for patient-specific internal emitter therapy. Medical Physics
24:1163-1172, 1997 (Exhibit 91). cited by applicant .
Scott AM et al., Image registration of SPECT and CT images using an
external fiduciary band and three-dimensional surface fitting in
metastatic thyroid cancer. J Nucl Med 36:100-103, 1995 (Exhibit
92). cited by applicant .
Sgouros G et al., Three-dimensional dosimetry for
radioimmunotherapy treatment planning. J Nucl Med 34:1595-1601,
1993 (Exhibit 93). cited by applicant .
Arndt CA et al., Common musculoskeletal tumors of childhood and
adolescence. N Engl J Med. 1999;341:342-52 (Exhibit 94). cited by
applicant .
West DC et al., Detection of circulating tumor cells in patients
with Ewing's sarcoma and peripheral primitive neuroectodermal
tumor. J Clin Oncol. 1997;15:583-8. (Exhibit 95). cited by
applicant .
de Alava E et al., Ewing family tumors: potential prognostic value
of reverse-transcriptase polymerase chain reaction detection of
minimal residual disease in peripheral blood samples. Diagn Mol
Pathol. 1998;7:152-7. (Exhibit 96). cited by applicant .
Toretsky JA et al., Detection of (11;22) (q24;q12)
translocation-bearing cells in peripheral blood progenitor cells of
patients with Ewing's sarcoma family of tumors. J Natl Cancer Inst.
1995;87:385-6. (Exhibit 97). cited by applicant .
Burdach S et al., Myeloablative radiochemotherapy and hematopoietic
stem-cell rescue in poor-prognosis Ewing's sarcoma. J Clin Oncol.
1993;11:1482-8. (Exhibit 98). cited by applicant .
Chan KW et al., High-dose sequential chemotherapy and autologous
stem cell reinfusion in advanced pediatric solid tumors. Bone
Marrow Transplant. 1997;20:1039-43. (Exhibit 99). cited by
applicant .
Fischmeister G et al., Low incidence of molecular evidence for
tumour in PBPC harvests from patients with high risk Ewing tumours.
Bone Marrow Transplant. 1999;24:405-9. (Exhibit 100). cited by
applicant .
Horowitz ME et al., Total-body irradiation and autologous bone
marrow transplant in the treatment of high-risk Ewing's sarcoma and
rhabdomyosarcoma. J Clin Oncol. 1993;11:1911-8. (Exhibit 101).
cited by applicant .
Perentesis J et al., Autologous stem cell transplantation for
high-risk pediatric solid tumors. Bone Marrow Transplant.
1999;24:609-15. (Exhibit 102). cited by applicant .
Chirgwin JM et al., Isolation of biologically active ribonucleic
acid from sources enriched in ribonuclease. Biochemistry.
1979;18:5294-9. (Exhibit 103). cited by applicant .
Mackall CL et al., Pathways of T-cell regeneration in mice and
humans: implications for bone marrow transplantation and
immunotherapy. Immunol Rev. 1997;157:61-72. (Exhibit 104). cited by
applicant .
Vogel W et al., Clinical applications of CD34(+) peripheral blood
progenitor cells (PBPC). Stem Cells. 2000;18:87-92. (Exhibit 105).
cited by applicant .
Dyson PG et al., CD34+ selection of autologous peripheral blood
stem cells for transplantation following sequential cycles of
high-dose therapy and mobilization in multiple myeloma [In Process
Citation]. Bone Marrow Transplant. 2000;25:1175-84. (Exhibit 106).
cited by applicant .
Emig M et al., Accurate and rapid analysis of residual disease in
patients with CML using specific fluorescent hybridization probes
for real time quantitative RT-PCR. Leukemia. 1999;13:1825-32.
(Exhibit 107). cited by applicant .
Mensink E et al., Quantitation of minimal residual disease in
Philadelphia chromosome positive chronic myeloid leukaemia patients
using real-time quantitative RT-PCR. Br J Haematol.
1998;102:768-74. (Exhibit 108). cited by applicant .
Pongers-Willemse MJ et al., Real-time quantitative PCR for the
detection of minimal residual disease in acute lymphoblastic
leukemia using junctional region specific TaqMan probes. Leukemia.
1998;12:2006-14. (Exhibit 109). cited by applicant .
Branford S et al., Monitoring chronic myeloid leukaemia therapy by
real-time quantitative PCR in blood is a reliable alternative to
bone marrow cytogenetics. Br J Haematol. 1999;107:587-99. (Exhibit
110). cited by applicant .
Chang H.R. et al., Expression of disialogangliosides G.sub.D2 and
G.sub.D3 on human soft tissue sarcomas. Cancer 70: 633-8, (1992)
(Exhibit 111). cited by applicant .
Froberg, K. et al., Intra-abdominal desmoplastic small round cell
tumor: immunohistochemical evidence for up-regulation of autocrine
and paracrine growth factors. Ann Clin Lab Sci 29: 78-85, 1999
(Exhibit 112). cited by applicant .
Heiner, J.P. et al., Localization of G.sub.D2-specific monoclonal
antibody 3F8 in human osteosarcoma. Cancer Res. 47: 5377-81 (1987)
(Exhibit 113). cited by applicant .
Kushner, B.H. et al., Desmoplastic small round-cell tumor:
prolonged progression-free survival with aggressive multimodality
therapy. J.Clin. Oncol. 14: 1526-31, (1996) (Exhibit 114). cited by
applicant .
Ladanyi, M. et al., Fusion of the EWS and WT1 genes in the
desmoplastic small round cell tumor. Cancer Res. 54: 2837-40,
(1994) (Exhibit 115). cited by applicant .
Gerald, W.L. et al., Intrabdominal desmoplastic small round cell
tumor. Report of 19 cases of a distinctive type of high-grade
polyphenotypic malignancy affecting young individuals. Am. L. Surg.
Pathol. 15, 499-513, (1991) (Exhibit 116). cited by applicant .
Gerald, W.L. et al., Clinical pathologic, and molecular spectrum of
tumors associated with t(11;22) (p13;q12): desmoplastic small
round-cell tumor and its variants. J. Clin. Oncol., 16: 3028-36,
(1998) (Exhibit 117). cited by applicant .
Ordonez, N.G. et al., Intra-abdominal desmoplastic small cell
tumor: a light microscopic, immunocytochemical, ultrastructural,
and flow cytometric study. Hum. Pathol. 24, 850-65, (1993) (Exhibit
118). cited by applicant .
Ordonez, N.G. et al., Desmoplastic small round cell tumor: II: an
ultrastructural and immunohistochemical study with emphasis on new
immunohistochemical markers. Am. J. Surg. Pathol. 22: 1314-27,
(1998) (Exhibit 119). cited by applicant .
Stancovski, I. et al., Targeting of T lymphocytes to
Neu/HERe-expressing cells using chimeric single chain Fv receptors.
J Immunol, 151: 6577-6582, 1993. (Exhibit 146). cited by applicant
.
Moritz, D. et al., Cytotoxic T lymphocytes with a grafted
recognition specificity for ERBB2-expressing tumor cells. Proc.
Natl Acad Sci, USA, 91: 4318-4322, 1994. (Exhibit 147). cited by
applicant .
Wels, W. et al., Biotechnological and gene therapeutic strategies
in cancer treatment. Gene, 159: 73-80, 1995. (Exhibit 148). cited
by applicant .
Eshhar, Z. et al., Functional expression of chimeric receptor genes
in human T cells. J Immunol Methods, 248: 67-76, 2001. (Exhibit
149). cited by applicant .
Wei, M. X. et al., Experimental tumor therapy in mice using the
cyclophosphamide-activating cytochrome P450 2B1 gene. Hum Gene
Ther, 5: 969, 1994. (Exhibit 150). cited by applicant .
Weijtens, M. E. et al., Single chain Ig/gamma gene-redirected human
T lymphocytes produce cytokines, specifically lyse tumor cells, and
recycle lytic capacity. J Immunol, 157: 836-843, 1996. (Exhibit
151). cited by applicant .
Finney, H. M. et al., Chimeric receptors providing both primary and
costimulatory signaling in T cells from a single gene product. J
Immunol, 161: 2791-2797, 1998. (Exhibit 152). cited by applicant
.
Koehne, G. et al., Rapid selection of antigen-specific T
lymphocytes by retroviral transduction. Blood, 96: 109-117, 2000.
(Exhibit 153). cited by applicant .
Bunnell, B. A. et al., High-efficiency retroviral-mediated gene
transfer into human nonhuman primate peripheral blood lymphocytes.
Proceeds of the National Academy of Science,USA, 92: 7739-7743,
1995. (Exhibit 154). cited by applicant .
Miller, A. D. et al., Construction and properties of retrovirus
packaging cells based on gibbon ape leukemia virus. J Virol, 1991:
2220-2224, 1991. (Exhibit 155). cited by applicant .
Lam, J. S. et al., Improved gene transfer into human lymphocytes
using retroviruses with gibbon ape leukemia virus envelope. Hum
Gene Ther, 7: 1415-1422, 1996. (Exhibit 156). cited by applicant
.
Bonini, C. et al., HSV-TK gene transfer into donor lymphocytes for
control of allogeneic graft-versus-leukemia. Science, 276:
1719-1723, 1997. (Exhibit 157). cited by applicant .
Pollok, K. E. et al., High-efficiency gene transfer into normal and
adenosine deaminase-deficient T lymphocytes is mediated by
transduction on recombinant fibronectin fragments. J Virol, 72:
4882-4892, 1998. (Exhibit 158). cited by applicant .
Galea-Lauri, J. et al., Expression of a variant of CD28 on a
subpopulation of human NK cells: implications for B7-mediated
stimulation of NK cells. J Immunol, 163: 62-70, 1999. (Exhibit
159). cited by applicant .
Patel, S. D. et al., Impact of chimeric immune receptor
extracellular protein domains on T cell function. Gene Therapy, 6:
412-419, 1999. (Exhibit 160). cited by applicant .
Fitzer-Attas, C. J. et al., Harnessing Syk familytyrosine kinases
as signaling domains for chimeric single chain of the variable
domain receptors: optional design for T cell activation. J Immunol,
160: 145-154, 1998. (Exhibit 161). cited by applicant .
Varez-Vallina, L. et al., Efficient discrimination between
different densities of target antigen by tetracycline-regulatable T
bodies. Hum Gene Ther, 10: 559-563, 1999. (Exhibit 162). cited by
applicant .
Yee, C. et al., In vivo tracking of tumor-specific T cells. Curr
Opin Immunol, 13: 141-146, 2001. (Exhibit 163). cited by applicant
.
Xiaoning, R. T. et al., Rapid death of adoptively transferred T
cells in acquired immunodeficiency syndrome. Blood, 93: 1506-1510,
1999. (Exhibit 164). cited by applicant .
Riddell, S. R. et al., T-cell mediated rejection of gene-modified
HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nat
Med, 2: 216-223, 1996. (Exhibit 165). cited by applicant .
Crist W et al., The Third Intergroup Rhabdomyosarcoma Study J Clin
Oncol 13:610-30, 1995 (Exhibit 166). cited by applicant .
Maurer HM et al., The Intergroup Rhabdomyosarcoma Study--II. Cancer
71:1904-22, 1993 (Exhibit 167). cited by applicant .
Weigel BJ et al., Role of high-dose chemotherapy with hematopoietic
stem cell rescue in the treatment of metastatic or recurrent
rhabdomyosarcoma. J Pediatr Hematol Oncol 23:272-276, 2001 (Exhibit
168). cited by applicant .
Kramer K et al., Targeted radioimmunotherapy for leptomeningeal
cancer using (131)I-3F8. Med Pediatr Oncol 35:716-8, 2000 (Exhibit
169). cited by applicant .
Kumar S et al., Myogenin is a specific marker for rhabdomyosarcoma:
an immunohistochemical study in paraffin embedded tissues. Mod
Pathol 13: 988-93, 2000 (Exhibit 170). cited by applicant .
Gattenloehner S et al., The fetal form of the acetylcholine
receptor distinguishes rhabdomyosarcomas from other childhood
tumors. Am J Pathol. 152:437-44, 1998 (Exhibit 171). cited by
applicant .
Truong LD et al., A study of 584 cases and review of the literature
Am J Clin Pathol 93:305-14, 1990 (Exhibit 172). cited by applicant
.
Qualman SJ et al., Intergroup Rhabdomyosarcoma Study: update for
pathologists Pediatr Dev Pathol 1:550-61, 1998 (Exhibit 173). cited
by applicant .
Strother DR et al., Expression of the 5.1 H11 antigen, a fetal
muscle surface antigen, in normal and neoplastic tissue. Arch
Pathol Lab Med 114:593-596, 1990 (Exhibit 174). cited by applicant
.
Merino ME et al., Immunomagnetic purging of ewing's sarcoma from
blood and bone marrow: quantitation by real-time polymerase chain
reaction. J Clin Oncol 19:3649-3659, 2001 (Exhibit 175). cited by
applicant .
International Search Report, Apr. 2, 2002 International Search
Report from Patent Cooperation Treaty for International Patent
Application Uses of Monoclonal Antibody 8H9 for Sloan-Kettering
Institute for Cancer Research, et al. International Filing Date
Oct. 18, 2001, claiming benefit of U.S. Appl. No. 60/241,344, filed
Oct. 18, 2000, and U.S. Appl. No. 60/330,396, filed Oct. 17, 2001.
(Exhibit 1). cited by applicant .
Juhl, et al., Additive Cytotoxicity of Different Monoclonal
Antibody-Cobra Venom Factor Conjugates for Human Neuroblastoma
Cells, Immunobiology, Nov. 1997, vol. 197, pp. 444-459 (Exhibit 2).
cited by applicant .
Modak, et al., Radioimmunotargeting to Human Rhabdomyosarcoma (RMS)
using Monoclonal Antibody (MOAB) 8H9, Proceedings of the American
Association for Cancer Research Annual Meeting, Mar. 2000 vol. 41
pp. 724, Abstract 4600. (Exhibit 3). cited by applicant .
Xu, et al., Targeting and therapy of carcinoembryonic
antigen-expressing tumors in transgenic mice with an
antibody-interleukin 2 fusion protein, Cancer Res Aug. 15, 2000,
vol. 60. No. 16, pp. 4475-4484, abstract only. (Exhibit 4). cited
by applicant .
Pegram, M. D., Slamon, D. J., Combination therapy with trastuzumab
(Herceptin) and cisplatin for chemoresistant metastatic breast
cancer: evidence for receptor-enhanced chemosensitivity. Sem.
Oncol., 26:89-95, 1999. (Exhibit 5). cited by applicant .
Bigner, D. D., et al., Iodine-131-labeled antitenascin monoclonal
antibody 81C6 treatment of patients with recurrent malignant
gliomas:phase I trial results. Journal Clincal Oncology,
16:2202-2212, 1998. (Exhibit 6). cited by applicant .
Bruland, O. et al., New monoclonal antibodies specific for human
sarcomas. Int J Cancer, 15:27-31, 1986. (Exhibit 7). cited by
applicant .
Wang, N. P. et al. Expression of myogenic regulatory
proteins(myogenin and MyoD1) in small blue round cell tumors of
childhood. Am. J. Pathol., 147:1799-1810, 1995. (Exhibit 8). cited
by applicant .
Bigner, D. D. et al., Phase I studies of treatment of malignant
gliomas and neoplastic meningitis with 131 I radiolabeled
monoclonal antibodies anti-tenascin 81C6 and anti-chondroitin
proteoglycan sulfate Mel-14 (ab')2--a preliminary report. J Neuro
Oncol, 24:109-122, 1995. (Exhibit 9). cited by applicant .
Mariani, G. et al., A pilot pharmacokinetic and immunoscintigraphic
study with the technetium-99m-labeled monoclonal antibody BC-1
directed against oncofetal fibronectin in patients with brain
tumors. Cancer Supplement, 80:2484-2489, 1997. (Exhibit 10). cited
by applicant .
DiMaggio JJ et al., Monoclonal antibody therapy of cancer. In:
Pinedo HM, Chabner BA, Longo, DL, (eds.): Cancer Chemotherapy and
Biological Response Modifiers, Annual 11, Elsevier Science
Publishers B.V., (Biomedical Division), 1990, pp. 177-203 (Exhibit
11). cited by applicant .
Schlom J., Monoclonal Antibodies in cancer therapy: Basic
principles. In: DeVita VT, Hellman S, Rosenberg SA, (eds.):
Biologic therapy of cancer, 2nd ed. Philadelphia, J.B.Lippincott
Co, 1995, pp. 507-520 (Exhibit 12). cited by applicant .
Lode HN. et al., Immunocytokines: A promising approach to cancer
immunotherapy. Pharmacology Therapeutics 80:277-292, 1998 (Exhibit
13). cited by applicant .
Erikson HP. et al., Hexabrachion protein (tenascin, cytotactin,
brachionectin) in connective tissues, embryonic tissues and tumors.
Adv Cell Biol 2:55-90, 1988 (Exhibit 14). cited by applicant .
Modak S. et al., Novel tumor-associated surface antigen: broad
distribution among neuroectodermal, mesenchymal and epithelial
tumors, with restricted distribution in normal tissues. Proceedings
of ASCO 17:449a, Abstract 1716 (Exhibit 15). cited by applicant
.
Cheung NK. et al., Treatment of advanced stage neuroblastoma. In:
Reghavan D, Scher HI, Leibel SA, Lange P, (eds.): Principles and
Practice of Genitourinary Oncology. Philadelphia, J.B. Lippincott
Company, 1997, pp. 1101-1111 (Exhibit 16). cited by applicant .
Brodeur G.M. et al., Neuroblastoma. In: Pizzo PA, Poplack DG,
(eds.): Principles and Practice of Pediatric Oncology, 3rd ed.
Philadelphia, J.B. Lippincott Company, 1997, pp. 761-797 chapter 2
(Exhibit 17). cited by applicant .
Cheung N.K.V. et al., Biological and molecular approaches to
diagnosis and treatment. section I. Principles of Immunotherapy.
In: Pizzo PA, Poplack DG, (eds.): Principles and Practice of
Pediatric Oncology, 3rd ed. ed. Philadelphia, J.B. Lippincott
Company, 1997, pp. 323-342 (Exhibit 18). cited by applicant .
Larson S.M. et al., Antibodies in cancer therapy: Radioisotope
conjugates. In: DeVita VT, Hellman S, Rosenberg SA, (eds.):
Biologic Therapy of Cancer, 2nd ed. Philadelphia, J.B. Lippincott
Co., 1995, pp. 534-552 (Exhibit 19). cited by applicant .
Reisfeld R.A. et al., Potential of genetically engineered
anti-ganglioside GD2 antibodies for cancer immunotherapy. In:
Progress in Brain Search (Svennerhol,L, Asbury,AK, Reisfeld,RA,
Sandhoff,K, Suzuki,K, Tettamani,G, Toffano,G, vol. 101. Cambridge,
UK, Elsevier Trends Journals, 1994, pp. 201-212 (Exhibit 20). cited
by applicant .
Cheung N.K.V. et al., Decay-accelerating factor protects human
tumor cells from complement-mediated cytotoxicity in vitro. J Clin
Invest 81:1122-1128, 1988 (Exhibit 21). cited by applicant .
Murray J.L. et al., Phase I trial of murine monoclonal antibody
14G2a administered by prolonged intravenous infusion in patients
with neuroectodermal tumors. J Biol Resp Modif 1991 (Soc. Biol.
Therapy Meeting Abstract 1991.) Journal of Clinical Oncology, vol.
12, No. 1 (Jan. 1994); pp. 184-193 (Exhibit 22). cited by applicant
.
Ugur O. et al., Comparison of the targeting characteristics of
various radioimmunoconjugates for radioimmunotherapy of
neuroblastoma: Dosimetry calculations incorporating cross-organ
beta doses. Nucl Med Biol 23:1-8, 1996 (Exhibit 23). cited by
applicant .
Saleh M.N. et al., Phase I trial of the murine monoclonal anti-GD2
antibody 14G2a in metastatic melanoma. Cancer Research 52,
4342-4347, Aug. 15, 1992 (Exhibit 24). cited by applicant .
Handgretinger R. et al., A phase I study of human-mouse chimeric
antiganglioside GD2 antibody ch14.18 in patients with
neuroblastoma. Eur J Cancer 31:261-267, 1995 (Exhibit 25). cited by
applicant .
Cheung N.K.V. et al., 3F8 monoclonal antibody treatment of patients
with stage IV neuroblastoma: a phase II study. In: Evans AE,
Guillio JD, Biedler JL, et al, (eds.): Advances in Neuroblastoma
Research, vol. 4. New York, Wiley Liss, 1994, pp. 319-329 (Exhibit
26). cited by applicant .
Yu A.L. et al., Phase I clinical trial of ch14.18 in patients with
refractory neuroblastoma. Proc Am Soc Clin Oncol 10:318, 1991,
Abstract 1118 (Exhibit 27). cited by applicant .
Cheung N.K., Biological and Molecular Approaches to Diagnosis and
Treatment. Immunotherapy. In: Pizzo PA, Poplack DG, (eds.):
Principles and Practice of Pediatric Oncology, 2nd ed.
Philadelphia, J.B.Lippincott Company, 1992, pp. 357-370 (Exhibit
28). cited by applicant .
Cheung N.K.V. et al., 3F8 monoclonal antibody treatment of patients
with stage IV neuroblastoma: A phase II Study. Int J Oncol
12:1299-1306, 1998 (Exhibit 29). cited by applicant .
Cheung N.K. et al., Phase I study of radioimmunotherapy of
neuroblastoma using iodine 131 labeled 3F8. In: Prog. Clin. Biol.
Res: Advances in Neuroblastoma Research 4. New York, Wiley Liss,
1994, pp. 329 (Exhibit 30). cited by applicant .
Kramer K. et al., Pharmacokinetics and acute toxicology of
intraventricular .sup.131I-monoclonal antibody targeting
disialoganglioside in non-human primates. J Neuro Oncol 1996
(Exhibit 31). cited by applicant .
Saleh M.N. et al., phase I trial of chimeric anti-GD2 monoclonal
antibody C14.18 in patients with metastatic melanoma. Hum. Antibod.
Hybridomas, 1992, vol. 3, January (Exhibit 32). cited by applicant
.
Cheung I.Y. et al., Induction of Ab3' following anti-GD2 monoclonal
antibody 3F8 therapy predicts survival among patients (pts) with
advanced neuroblastoma. Proc Am Assoc Cancer Res 40:574, 1999,
Abstract 3787 (Exhibit 33). cited by applicant .
Chen S. et al., Surface antigen expression and complement
susceptibility of differentiated neuroblastoma clones. Am J Pathol
In press:, 1999 (Exhibit 34). cited by applicant .
Sgouros G. et al., Hematologic toxicity in radioimmunotherapy: An
evaluation of different predictive measures. J Nucl Med 37:43P-44P,
1996, Abstract 165 (Exhibit 35). cited by applicant .
Sgouros G. et al., Treatment planning for internal emitter therapy:
methods, applications and clinical implications. 1996 (Exhibit 36).
cited by applicant .
Sgouros G. et al., Yttrium-90 biodisribution by yttrium-87 imaging:
a feasibility analysis. Medical Physics 25(8), Aug. 1998 (Exhibit
37). cited by applicant .
Meyer C.R. et al., Demonstration of accuracy and clinical
versatility of mutual information for automatic multimodality image
fusion using affine and thin-plate spline warped geometric
deformations. Medical Image Analysis 1:195-206, 1997 (Exhibit 38).
cited by applicant .
Burdach S. et al., Myeloablative therapy, stem cell rescue and gene
transfer in advanced Ewing tumors. Bone Marrow Transplant. 1996;18
Suppl 1:S67-8. (Exhibit 39). cited by applicant .
Ladenstein R. et al., Impact of megatherapy in children with
high-risk Ewing's tumours in complete remission: a report from the
EBMT Solid Tumour Registry [published erratum appears in Bone
Marrow Transplant Sep. 1996;18(3):675]. Bone Marrow Transplant.
1995;15:697-705. (Exhibit 40). cited by applicant .
Ladenstein R. et al., Autologous stem cell transplantation for
solid tumors in children. Curr Opin Pediatr. 1997; 9:55-69.
(Exhibit 41). cited by applicant .
Laws H.J. et al., Multimodality diagnostics and megatherapy in poor
prognosis Ewing's tumor patients. A single-center report.
Strahlenther Onkol. 1999;175:488-94. (Exhibit 42). cited by
applicant .
Pape H. et al., Radiotherapy and high-dose chemotherapy in advanced
Ewing's tumors. Strahlenther Onkol. 1999; 175:484-7. (Exhibit 43).
cited by applicant .
Pession A. et al., Phase I study of high-dose thiotepa with
busulfan, etoposide, and autologous stem cell support in children
with disseminated solid tumors. Med Pediatr Oncol. 1999; 33:450-4.
(Exhibit 44). cited by applicant .
Stewart D.A. et al., High-dose melphalan +/- total body irradiation
and autogolous hematopoietic stem cell rescue for adult patients
with Ewing's sarcoma or peripheral neuroectodermal tumor. Bone
Marrow Transplant. 1996; 18:315-8. (Exhibit 45). cited by applicant
.
Rill D.R. et al., Direct demostration that autologous bone marrow
transplantation for solid tumors can return a multiplicity of
tumorigenic cells. Blood. 1994; 84:380-3. (Exhibit 46). cited by
applicant .
Brenner M.K. et al., Gene-marking to trace origin of relapse after
autologous bone-marrow transplantation. Lancet. 1993; 341:85-6.
(Exhibit 47). cited by applicant .
Mackall C. et al., Combined Immune Reconstitution/Tumor Vaccination
to induce anti-tumor immune responses in the setting of minimal
residual neoplastic disease [abstract]. Blood. 1999; 94:133a,
Abstract 586 (Exhibit 48). cited by applicant .
Quinones R.R. et al., Extended-cycle elutriation to adjust T-cell
content in HLA-disparate bone marrow transplantation. Blood. 1993;
82:307-17. (Exhibit 49). cited by applicant .
Kontny H.U. et al., Simultaneous expression of Fas and
nonfunctional Fas ligand in Ewing's sarcoma. Cancer Res. 1998;
58:5842-9. (Exhibit 50). cited by applicant .
de Wynter E.A. et al., Comparison of purity and enrichment of CD34+
cells from bone marrow, umbilical cord and peripheral blood (primed
for apheresis) using five separatoin systems. Stem Cells. 1995;
13:524-32. (Exhibit 51). cited by applicant .
Dworzak M.N. et al., Flow cytometric assessment of human MIC2
expression in bone marrow, thymus, and peripheral blood. Blood.
1994; 83:415-25. (Exhibit 52). cited by applicant .
De Leij, et al., SCLC-cluster-2 antibodies detect the
pancarcinoma/epithelial glycoprotein E GP-2 (supplement) Int. J.
Cancer 8: 60-3, 1994 (Exhibit 53). cited by applicant .
Willian L. et al., Intra-abdominal desmoplastic small round-cell
tumors: report of 19 cases of distinctive type of high-grade
polyphenotypic malignancy affecting young individuals Am. J. Surg.
Pathol. 15(6): 499-513, (1991) (Exhibit 54). cited by applicant
.
Lee, S.B. et al., The EWS-WT1 translocation product induces PDGFA
in desmoplastic small round-cell tumour. Nat Genet 17, 309-13, 1997
(Exhibit 55). cited by applicant .
Daniel, P. T. et al., Costimulatory signals through B7.1/CD28
prevent T cell apoptosis during target cell lysis. J Immunol, 159:
3808-3815, 1997. (Exhibit 60). cited by applicant .
Hwu, P. et al., Lysis of ovarian cancer cells by human lymphocytes
redirected with a chimeric gene comosed of an antibody variable
region and the Fc-receptor gamma-chain. J. Exp. Med., 178: 361-369,
1993. (Exhibit 61). cited by applicant .
Rosenberg, S. A., Cell transfer therapy: clinical applications. In:
V. T. J. DeVita, S. Hellman, and S. A. Rosenberg (eds.), Biologic
therapy of cancer, second edition, pp. 487-506. Philadelphia:
J.B.Lippincott Company, 1995. (Exhibit 62). cited by applicant
.
Yang, A.-G. et al., A new class of antigen-specific killer cells.
Nat Biotechnol, 15: 46-51, 1997. (Exhibit 63). cited by applicant
.
Culver, K. W. et al., In vivo gene transfer with retroviral
vector-producer cells for treatment of experimental brain tumors.
Science, 256: 1550, 1992. (Exhibit 64). cited by applicant .
Jensen, M. et al., CD20 is a molecular target for scFvFc: receptor
redirected T cells: implications for cellular immunotherapy of
CD20+ malignancy. Biol Blood Marrow Transplant, 4: 75-83, 1998.
(Exhibit 65). cited by applicant .
Eshhar, Z. et al., Tyrosine kinase chimeras for antigen-selective
T-body therapy. Adv Drug Deliv Rev, 31: 171-182, 1998. (Exhibit
66). cited by applicant .
Valitutti, S. et al., Serial triggering of TCRs: a basis for the
sensitivity and specificity of antigen recognition. Immunology
Today, 18: 299-304, 1997. (Exhibit 67). cited by applicant .
Ruymann F.B. et al., Progress in the diagnosis and treatment of
rhabdomyosarcoma and related soft tissue sarcomas. Cancer Invest
18:223-241, 2000 (Exhibit 68). cited by applicant .
Cheung N.K. et al., Targeting of ganglioside GD2 monoclonal
antibody to neuroblastoma J Nuc Med 28:1577-83, 1987 (Exhibit 69).
cited by applicant .
Thomson B. et al., RT-PCR evaluation of peripheral blood, bone
marrow and peripheral blood stem cells in children and adolescents
undergoing VACIME chemotherapy for Ewing's sarcoma and alveolar
rhabdomyosarcoma. Bone Marrow Transplant 24:527-33, 1999 (Exhibit
70). cited by applicant .
Athale U.H. et al., Use of Reverse Transcriptase Polymerase Chain
Reaction for Diagnosis and Staging of Alveolar Rhabdomyosarcoma,
Ewing Sarcoma Family of Tumors, and Desmoplastic Small Round Cell
Tumor. Am J Pediatr Hematol Oncol 23(2):99-104, 2001 (Exhibit 71).
cited by applicant .
Mackall C. et al., Targeting tumor specific translocations in
sarcomas in perdiatric patients for immunotherapy. Clin Orthop.
373:25-31, 2000 (Exhibit 72). cited by applicant .
Gruchala A. et al., Rhabdomyosarcoma. Morphologic,
immunohistochemical, and DNA study. Gen Diagn Pathol 1142:175-84,
1997 (Exhibit 73). cited by applicant .
Kalebic T. et al., In vivo treatment with antibody against IGF-1
receptor suppresses growth of human rhabdomyosarcoma and
down-regulates p34.sup.cdc2. Cancer Res 54:5531-4, 1994 (Exhibit
74). cited by applicant .
International Search Report for PCT/US03/07004, filed Mar. 6, 2003
for Sloan-Kettering Institute for Cancer Research et al., dated
Jun. 7, 2005. cited by applicant .
Supplemental European Search Report for EP 01 98 3999, filed May
16, 2003, for Sloan-Kettering Institute for Cancer Research et al.,
dated Jul. 26, 2005. cited by applicant .
Modak, et al., "Disialoganglioside GD2 and antigen 8H9: Potential
targets for antibody-based immunotherapy against desmoplastic small
round cell tumor (DSRCT) and rhabdomyosarcoma (RMS)", Proceedings
of the American Assosciation for Cancer Search Annual Meeting, vol.
40, Mar. 1999, p. 474. cited by applicant .
Anja Krause, Hong Fen Guo, Jean-Baptiste Latouche, Cuiwen Tan,
Nai-Kong V. Cheung, Michael Sadelain, Antigen-Dependent CD28
Signaling Selectively Enhances Survival and Proliferation in
Genetically Modified Activated Human Primary T Lymphocytes, J. Exp.
Med, vol. 188, No. 4, pp. 619-626 (1998). cited by applicant .
Cheung Nai-Kong V. et al., "Anti-idiotypic Antibody Facilitates
scFv Chimeric Immune Receptor Gene Transduction and Clonal
Expansion of Human Lymphocytes for Tumor Therapy", Hybridoma and
Hybridomics vol. 22, No. 4, pp. 209-218 (2003). cited by applicant
.
Cheung Nai-Kong V. et al., "Anti-idiotypic Antibody as the
Surrogate Antigen for Cloning scFv and its Fusion Proteins",
Hybridoma and Hybridomics vol. 21, No. 6, pp. 433-443 (2002). cited
by applicant .
Cheung Nai-Kong V., "Monoclonal Antibody-based Therapy for
Neuroblastoma", Current Oncology Reports, vol. 2, No. 6, pp.
547-553 (2000). cited by applicant .
International Patent Publication No. WO 02/32375 for
Sloan-Kettering Institute for Cancer Research, Filed Oct. 18, 2001
for "Uses of Monoclonal Antibody 8H9". cited by applicant .
Modak S., Guo H.F., Humm J.L., Smith-Jones P.M., Larson S.M.,
Cheung N.K., "Radioimmunotargeting of Human Rhabdomyosarcoma Using
Monoclonal Antibody 8H9", Cancer Biother Radiopharm, 20(5):534-48
(2005). cited by applicant .
Modak S., Guo H.F., Humm J., Larson S.M., Cheung N.K., "Novel Tumor
Target for Antibody-based Therapy of Rhabdomyosarcoma and Other
Pediatric Solid Tumors", Journal of Pediatric Hematology/Oncology
22(4) (2000). cited by applicant .
Modak S., Gultekin S.H., Kramer K., Guo H.F., Rosenfeld M.R.,
Ladanyi M., Larson S.M., Cheung Nai Kong V., "Novel
Tumor-associated Surface Antigen: Broad Distribution among
Neuroectodermal Mesenchymal and Epithelial Tumors with Restructured
Distribution in Normal Tissues", Proceedings of ASCO vol. 17
(1998). cited by applicant .
Modak S., Kramer K., Gultekin S.H., Guo H.F., Larson S.M., Cheung
N.K., "Monoclonal Antibody 8H9: Specific for a Novel Tumor Antigen
on Human Neuroblastoma", Medical and Pediatric Oncology, vol. 35,
No. 6 (2000). cited by applicant .
Queen, et al. "A humanized antibody that binds to the interleukin
receptor", Proc. Natl. Acad. Sci USA, vol. 86, pp. 10029-10033,
Dec. 1989. cited by applicant .
PCT International Preliminary Examination Report for
Sloan-Kettering Institute for Cancer Research, Int'l Application
No. PCT/US01/32565, Filed Oct. 18, 2001, Dated Apr. 27, 2006. cited
by applicant .
PCT Written Opinion of the International Preliminary Examining
Authority for Sloan-Kettering Institute for Cancer Research, Int'l
Application No. PCT/US01132565, Filed Oct. 18, 2001, Dated Oct. 25,
2005. cited by applicant .
PCT Application Publication No. PCT/US97/04427 for Sloan-Kettering
Institute for Cancer Research et al., Filed Mar. 20, 1997 for
"Single Chain FV Constructs of Anti-ganglioside GD", Published with
International Search Report. cited by applicant .
European Patent Office Supplementary Search Report for
Sloan-Kettering Institute for the Cancer Research, Int'l No.
PCT/US03/07004, Filed Mar. 4, 2003 Dated Oct. 25, 2005. cited by
applicant .
PCT Notification of Transmittal of International Search Report,
Apr. 2, 2002, for Sloan-Kettering Institute for Cancer Research,
Int'l App'l No. PCT/US01/32565, filed Oct. 18, 2001. cited by
applicant .
PCT Notification of Transmittal of the International Search Report,
Dec. 22, 2003, for Sloan-Kettering Institute for Cancer Research,
Int'l App'l No. PCT/US02/33331, filed Oct. 17, 2002. cited by
applicant .
Supplementary European Search Report, Oct. 12, 2005, for
Sloan-Kettering Institute for Cancer Research, European App'l No.
EP 02 80 1782, filed Apr. 23, 2004. cited by applicant .
Supplementary European Search Report, Jul. 26, 2005, for
Sloan-Kettering Institute for Cancer Research, European App'l No.
EP 01 98 3999 filed May 16, 2003. cited by applicant .
Supplementary European Search Report, Oct. 14, 2005, for
Sloan-Kettering Institute for Cancer Research, European App'l No.
EP 03 71 6369 filed Oct. 8, 2004. cited by applicant .
EPO Communication, Jan. 16, 2006, Sloan-Kettering Institute for
Cancer Research, European App'l No. EP 03 716 369.8 filed Oct. 8,
2004. cited by applicant .
Alvarez-Vallina et al., 1996, "Antigen-specific targeting of CD28
receptors," European Journal of Immunology, 26(10):2304-2309. cited
by applicant .
Baxevanis et al., 2004, "Targeting of tumor cells by lymphocytes
engineered to express chimeric receptor genes", Cancer Immunol
Immunother, 53:893-903. cited by applicant .
Botti et al., 1997, "Comparison of three different methods for
radiolabelling human activated T lymphocytes", Eur. J. Nucl. Med.
24:497-504. cited by applicant .
Curti, 1993, "Physical barriers to drug delivery in tumors", Crit.
Rev. in Oncology/Hematology, 14:29-39. cited by applicant .
Elliott et al., 1999, "SSTR2A is the dominant somatostatin receptor
subtype expressed by inflammatory cells, is widely expressed and
directly regulates T cell IFN-gamma release", Eur. J. Immunol.
29:2454-63. cited by applicant .
Fisher et al., 1989, "Tumor localization of adoptively transferred
indium-111 labeled tumor infiltrating lymphocytes in patients with
metastatic melanoma", J. Clin. Oncol. 7:250-261. cited by applicant
.
Gong et al.,1994, "Characterization of a human cell line (NK-92)
with phenotypical and functional characteristics of activated
natural killer cells", Leukemia, 8:652-8. cited by applicant .
Gura, T., 1998, "Systems for identifying new drugs are often
faulty", Science, 278:1041-1042. cited by applicant .
Heppeler et al., 1999, "Radiometal-labelled macrocyclic
chelator-derivatized somatostatin analogue with superb
tumour-targetting properties and potential for receptor-mediated
internal radiotherapy," Chemistry-A European Journal,
5(7)1974-1981. cited by applicant .
Heslop et al., 1997, "Adoptive cellular immunotherapy for EBV
lymphoproliferative diseases", Immunological Reviews 157:217-222.
cited by applicant .
Heslop et al., 1996, "Long-term restoration of immunity against
Epstein-barr virus infection by adoptive transfer of gene-modified
virus-specific T lymphocytes", Nature Med., 2:551-555. cited by
applicant .
Hombach, A. et al., 1998, "Isolation of Single Chain Antibody
Fragments with Specificity for Cell Surface Antigens by Phage
Display Utilizing Internal Image Anti-Idiotypic Antibodies", J.
Immunol. Methods, 218:53-61. cited by applicant .
Jain, R.K., 1994, "Barriers to drug delivery in solid tumors", Sci.
Am., 271:58-65. cited by applicant .
Koehne et al, 2003, "Serial in vivo imaging of the targeted
migration of human HSV-TK-transduced antigen-specific lymphocytes
", Nature Biotechnology, 21:405-413. cited by applicant .
Koehne et al., 2000, "Noninvasive imaging of human radiolabeled
antigen-specific donor T lymphocytes after adoptive immunotherapy
in SCID-mice", Blood, 96:516a, abstract #2222. cited by applicant
.
Koprowski et al., 1984, "Human anti-idiotype antibodies in cancer
patients: Is the modulation of the immune response beneficial for
the patient?", Proc. Natl, Acad. Sci, USA, 81:216-219. cited by
applicant .
Kramer et al., 1997, "Pharmacokinetics and acute toxicology of
intraventricular 131I-monoclonal antibody targeting
disialoganglioside in non-human primates," J. Neuro. Oncol.,
35:101-111. cited by applicant .
Kundra et al., 2002, "Noninvasive monitoring of somatostatin
receptor type 2 chimeric gene transfer", J. Nucl. Med. 43:406-12.
cited by applicant .
Lacerda et al., 1996, "Human Epstein-Barr virus (EBV)-specific
cytoxic T lymphocytes home preferentially to and induce selective
regressions of autologous EBV-induced B cell lymphoproliferations
in xenografted C.B-17 Scid/Scid mice", J. Exp. Med., 183:1215-1228.
cited by applicant .
Ma et al., 2002, "Genetically engineered T cell as adoptive
immunotherapy of cancer", in Giaccone G, Schilsky, R, Sondel, P.
(ed), Cancer Chemotherapy and Biological Response Modifiers.
Amsterdam, Elsevier Science B.V., chapter 15, pp. 315-341. cited by
applicant .
Maher et al., 2002, "Human T-lymphocyte cytoxity and proliferation
directed by a single chimeric TCRzeta/CD28 receptor", Nat.
Biotechnol. 20:70-5. cited by applicant .
Maki et al., 2001, "Factors regulating the cytoxic activity of the
human natural killer cell line, NK-92", J. Hematother. Stem Cell
Res. 10:369-83. cited by applicant .
McGuiness et al., 1999, "Anti-tumor activity of human T cells
expressing the CC49-zeta chimeric immune receptor", Human Gene
Therapy, 10:165-173. cited by applicant .
Modak, et al., 1999, "Disialoganglioside GD2 and antigen 8H9:
Potential targets for antibody-based immunotherapy against
desmoplastic small round cell tumor (DSRCT) and rhabdomyosarcoma
(RMS)", Proceedings of the American Association for Cancer Research
Annual Meeting, #3133. cited by applicant .
Papadopoulos et al., 1994, "Infusions of donor leukocytes to treat
Epstein-Barr virus-associated lymphoproliferative disorders after
allogeneic bone marrow transplantation", N. Engl. J. Med.,
330:1185-1191. cited by applicant .
Reinhold et al., 1999, "Specific lysis of melanoma cells by
receptor grafted T cells is enhanced by anti-idiotypic monoclonal
antibodies directed to scFv domain of the receptor", Journal of
Investigative Dermatology, 112: 744-750. cited by applicant .
Riddell et al., 1992, "Restoration of viral immunity in
immunodeficient humans by the adoptive transfer of T cell clones",
Science, 257:238-241. cited by applicant .
Rosenberg et al., 1988, "A progress report on the treatment of 157
patients with advanced cancer using lymphokine-activated killer
cells and interleukin-2 or high-dose interleukin-2 alone", N. Engl.
J. Med., 316: 889-897. cited by applicant .
Rossig et al., 2002, "Epstein-Barr virus=--specific human T
lymphocytes expressing antitumor chimeric T-cell receptors
potential for improved immunotherapy", Blood, 99:2009-16. cited by
applicant .
Rossig et al., 2001, "Targeting of G(D2)-positive tumor cells by
human T lymphocytes engineered to express chimeric T-cell receptor
genes", Int. J. Cancer, 94:228-36. cited by applicant .
Schlebusch H., et. al., 1997, "Production of a Single-Chain
Fragment of the Murine Anti-Idiotypic Antibody ACA125 as
Phage-Displayed and Soluble Antibody by Recombinant Phage Antibody
Technique", Hybridoma, 16(1):47-52. cited by applicant .
Smanik et al., 1996, "Cloning of the human sodium iodide
symporter", Biochem. Biophys. Res. Comm., 226:339-345. cited by
applicant .
Tonn et al., 2001, "Cellular immunotherapy of malignancies using
the clonal natural killer cell line NK-92", J. Hematother. Stem
Cell Res., 10:535-44. cited by applicant .
Tsutsumi et al.,1997, "Expression of somatostatin receptor subtype
2 mRNA in human lymphoid cells", Cell Immunol., 181:44-9. cited by
applicant .
Ugur et al., 2002, "ga-66 labeled somatostatin analogue
DOTA-DPhel-Tyr3-octreotide as a potential agent for positron
emission tomography imaging and receptor mediated internal
radiotherapy of somatostatin receptive positive tumors", Nucl. Med.
Biol. 29:147-57. cited by applicant .
Walter et al., 1995, "Reconstitution of cellular immunity against
cytomegalovirus in recipients of allogeneic bone marrow by transfer
of T-cell clones from the donor", N. Engl. J. Med., 333:1038-1044.
cited by applicant .
Zhang W., et. al., 2002, "Production and Characterization of Human
Monoclonal Anti-Idiotype Antibodies to Anti-dsDNA Antibodies",
Lupus, 11(6):362-369. cited by applicant .
Zinn et al., 2002, "Gamma camera dual imaging with a somatostatin
receptor and thymidine kinase after gene transfer with a
bicistronic adenovirus in mice", Radiology, 223:417-25. cited by
applicant .
U.S. Office Action, Jul. 26, 2006, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant .
U.S. Office Action, Dec. 27, 2007, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant .
U.S. Office Action, May 16, 2008, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant .
U.S. Office Action, Apr. 5, 2006, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Office Action, Sep. 7, 2006, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Office Action, Mar. 23, 2007, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Office Action, Jul. 10, 2007, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Office Action, Jan. 14, 2008, for Nai-Kong V. Cheuno, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Notice of Allowance, Oct. 30, 2008, for Nai-Kong V. Cheung,
U.S. Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant
.
U.S. Office Action, Sep. 22, 2004, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant .
U.S. Office Action, Aug. 24, 2004, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant .
U.S. Office Action, Nov. 17, 2005, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Advisory Action, Dec. 13, 2006, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Advisory Action, Jan. 22, 2007, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Office Action, Sep. 15, 2008, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
U.S. Office Action, Feb. 20, 2009, for Nai-Kong V. Cheung and
Hong-fen Guo, U.S. Appl. No. 10/273,762, filed Oct. 17, 2002. cited
by applicant .
Kawai et al., 1999, "Occurrence of ganglioside GD3 in neoplastic
astrocytes", Virchows Arch, 434:201-205. cited by applicant .
PCT International Search Report for the Government of the United
States of America as represented by the Secretary, Department of
Health and Human Services and Memorial Sloan-Kettering Cancer
Center, Nov. 22, 2004, Int'l Application No. PCT/US03/38227. cited
by applicant .
Australian Office Action, Mar. 24, 2009, for Ira Pastan, Australian
Application No. EP 2003298794. cited by applicant .
Canadian Office Action, Jul. 8, 2009, for Sloan-Kettering Institute
for Cancer Research, Canadian Application No. 2,423,843. cited by
applicant .
European Office Action, May 19, 2006, for Sloan-Kettering Institute
for Cancer Research, European Application No. 01 98 3999. cited by
applicant .
European Office Action, Jan. 26, 2007, for Sloan-Kettering
Institute for Cancer Research, European Application No. 02801782.
cited by applicant .
European Office Action, May 23, 2007, for Sloan-Kettering Institute
for Cancer Research, European Application No. 03 716 369.8. cited
by applicant .
European Office Action, Aug. 16, 2006, for Sloan-Kettering
Institute for Cancer Research, European Application No. 03 716
369.8. cited by applicant .
European Communmication (Interview Summary), Sep. 13, 2007, for
Sloan-Kettering Institute for Cancer Research, European Application
No. 03 716 369.8. cited by applicant .
European Office Action, Jun. 25, 2007, for Ira Pastan, European
Application No. EP 03 796 552.2. cited by applicant .
U.S. Office Action, Feb. 22, 2007, for Ira Pastan, U.S. Appl. No.
10/537,061, filed Jun. 1, 2005. cited by applicant .
U.S. Office Action, Aug. 14, 2007, for Ira Pastan, U.S. Appl. No.
10/537,061, filed Jun. 1, 2005. cited by applicant .
U.S. Office Action, Jan. 24, 2008, for Ira Pastan, U.S. Appl. No.
10/537,061, filed Jun. 1, 2005. cited by applicant .
Modak et al., 2002, "Disialoganglioside GD2 and a novel tumor
antigen: potential targets for immunotherapy of desmoplastic small
round cell tumor", Med. Pediatr. Oncol., 39:547-551. cited by
applicant .
Onda et al., 2004, "In vitro and in vivo cytoxic activities of
recombinant immunotoxin 8H9(Fv)-PE38 against breast cancer,
osteosarcoma, and neuroblastoma", Cancer Research, 64:1419-1424.
cited by applicant .
Luther et al., 2008, "Intraparenchymal and Intratumoral
Interstitial Infusion of Anti-Glioma Monoclonal Antibody 8H9",
Neurosurgery, 63:1166-1174. cited by applicant .
Xu et al., 2009, "MicroRNA miR-29 Modulates Expression of
Immunoinhibitory Molecule B7-H3: Potential Implications for Immune
Based Therapy of Human Solid Tumors", Cancer Research, 69(15).
cited by applicant .
European Office Action (FER), Mar. 30, 2006, for The Government of
the United States of America as represented by The Secretary,
Department of Health and Human Services and Memorial
Sloan-Kettering Cancer Center, European Application No. EP 03 796
552.2. cited by applicant .
Supplementary Partial European Search Report for The Government of
the United States of America as represented by The Secretary,
Department of Health and Human Services and Memorial
Sloan-Kettering Cancer Center, Feb. 3, 2006, European Application
No. EP 03 796 552.2. cited by applicant .
Cheung et al., 2004, "Single chain Fv-streptavidin substantially
improved therapeutic index in multi-step targeting directed at
disialoganglioside GD2", J. Nucl. Med., 867-877. cited by applicant
.
Murray et al., 1996, "Phase Ia/Ib trial of anti-GD2 chimeric
monoclonal antibody 14.18 (ch14.18) and recombinant human
granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in
metastatic melanoma", J. Immunother. Emphasis Tumor Immunol.,
19:206-17. cited by applicant .
U.S. Notice of Allowance, Sep. 23, 2009, for Nai-Kong V. Cheung,
U.S. Appl. No. 10/097,558, filed Mar. 8, 2002. cited by applicant
.
U.S. Decision Granting Petition Under 37 CFR 1.313(c)(2), Jul. 28,
2009, for Nai-Kong V. Cheung, U.S. Appl. No. 10/097,558, filed Mar.
8, 2002. cited by applicant .
U.S. Notice of Allowance, Sep. 25, 2009, for Nai-Kong V. Cheung,
U.S. Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant
.
U.S. Interview Summary, Aug. 11, 2009, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/273,762, filed Oct. 17, 2002. cited by applicant .
PCT Notification Concerning Transmittal of International
Preliminary Report on Patentability for Sloan-Kettering Institute
for Cancer Research et al., Sep. 22, 2009, Int'l Application No.
PCT/US2008/058030. cited by applicant .
U.S. Office Action, Dec. 13, 2006, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant .
U.S. Office Action, Apr. 2, 2007, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant .
U.S. Office Action, Jan. 17, 2007, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant .
U.S. Office Action, Sep. 24, 2008, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant .
U.S. Office Action, Mar. 20, 2008, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant .
U.S. Office Action, Mar. 2, 2009, for Nai-Kong V. Cheung, U.S.
Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant .
U.S. Notice of Allowance, Aug. 21, 2009, for Nai-Kong V. Cheung,
U.S. Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant
.
Canadian Office Action, Nov. 2, 2009, for Sloan-Kettering Institute
for Cancer Research, Canadian Application No. 2,463,017. cited by
applicant .
U.S. Notice of Allowance, Dec. 14, 2009, for Nai-Kong V. Cheung,
U.S. Appl. No. 10/505,658, filed Aug. 20, 2004. cited by applicant
.
Supplementary Partial European Search Report for Sloan-Kettering
Institute for Cancer Research, Mar. 1, 2010, European Application
No. EP 08 74 4263. cited by applicant .
European Result of Consultation, Mar. 12, 2010, for Sloan-Kettering
Institute for Cancer Research, for for EP 03 716 369.8, Filed Oct.
18, 2004, National Stage of PCT/US03/07004, Filed Mar. 6, 2003.
cited by applicant .
European Communication under Rule 71(3) EPC, Apr. 28, 2010, for
Sloan-Kettering Institute for Cancer Research, for for EP 03 716
369.8, Filed Oct. 18, 2004, National Stage of PCT/US03/07004, Filed
Mar. 6, 2003. cited by applicant .
European Office Action, Jul. 1, 2010, for Sloan-Kettering Institute
for Cancer Research, for EP 01 98 3999 , Filed May 16, 2003,
National Stage of PCT/US01/32565, Filed Oct. 18, 2001. cited by
applicant .
Canadian Office Action, Sep. 20, 2010, for Sloan-Kettering
Institute for Cancer Research, Canadian Application No. 2,423,843.
cited by applicant .
Canadian Office Action, Nov. 18, 2010, for Sloan-Kettering
Institute for Cancer Research, Canadian Application No. 2,478,082.
cited by applicant .
U.S. Office Action, Nov. 26, 2010, for Nai-Kong V. Cheung, U.S.
Appl. No. 12/709,848, filed Feb. 22, 2010. cited by applicant .
European Office Action, Nov. 15, 2010, for Sloan-Kettering
Institute for Cancer Research, for for EP 01 983 999.2, Filed May
16, 2003, National Stage of PCT/US01/32565, Filed Oct. 18, 2001.
cited by applicant .
Adams et al., 1993, "Highly specific in vivo tumor targeting by
monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain
Fv", Cancer Research, 53:4026-4034. cited by applicant .
Alt eta l., 1999, "Novel tetravalent and bispecific IgG-like
antibody molecules combining single-chain diabodies with the
immunoglobulin y1 Fc or CH3 region", FEBS Letters, 454:90-94. cited
by applicant .
Bird et al., 1988, "Single-chain antigen-binding proteins",
Science, 242:423-426. cited by applicant .
Brocks et al., 1997, "A TNF receptor antagonistic scFv, which is
not secreted in mammalian cells, is expressed as a soluble mono-
and bivalent scFv derivative in insect cells", Immunotechnology,
3:173-84. cited by applicant .
Burton, D.R. and Barbas III, C.G., 1994, "Human antibodies from
combinatorial libraries", Advances in Immunology, 57:191-280. cited
by applicant .
Cheung NK, 2007, "Therapeutic antibodies and immunologic
conjugates" in Abele MD, Armitage JO, Niederhuber JE, et al (eds):
Clinical Oncology (ed 4th). Philadelphia, Elsevier Churchill
Livingstone, Chapter 34, pp. 531-544. cited by applicant .
Cai, X. and Garen, A., 1995, "Anti-melanoma antibodies from
melanoma patients immunized with genetically modified autologous
tumor cells: selection of specific antibodies from single-chain Fv
fusion phage libraries", Proceedings of the National Academy of
Sciences of the United States of America, 92:6537-41. cited by
applicant .
George, A.J.T., Spooner, R.A. and Epenetos, A.A. (1994)
Applications of Monoclonal Antibodies in Clinical Oncology.
Immunology Today 15, 559-561. cited by applicant .
Ghetie et al., 1997, "Homodimerization of tumor-reactive monoclonal
antibodies markedly increases their ability to induce growth arrest
or apoptosis of tumor cells", Proceedings of the National Academy
of Sciences of the United States of America, 94:7509-14. cited by
applicant .
Huston et al., 1988, "Protein engineering of antibody binding
sites: recovery of specific activity in an anti-digoxin
single-chain Fv analogue produced in Escherichia coli", Proceedings
of the National Academy of Sciences of the United States of
America, 85:5879-83. cited by applicant .
Kato et al., 1995, "Mammalian expression of single chain variable
region fragments dimerized by Fc regions", Molecular Biology
Reports, 21:141-146. cited by applicant .
Kipriyanov et al., 1995, "Single-chain antibody streptavidin
fusions: tetrameric bifunctional scFv-complexes with biotin binding
activity and enhanced affinity to antigen", Human Antibodies
Hybridomas, 6:93-101. cited by applicant .
Kushner, B. H. and Cheung, N. K., 1992, "Absolute requirement of
CD11/CD18 adhesion molecules, FcRII and phosphatidylinositol-linked
FcRIII for monoclonal antibody-mediated neutrophil anti-human tumor
cytotoxicity", Blood, 79:1484-1490. cited by applicant .
Laemmli, U.K., 1970, "Cleavage of structural proteins during the
assembly of the head of bacteriophage T4", Nature, 227:680-85.
cited by applicant .
Lu, J. and Sloan, S.R., 1999, "An alternating selection strategy
for cloning phage display antibodies", Journal of Immunological
Methods, 228:109-119. cited by applicant .
Michael et al., 1996, "In vitro and in vivo characterisation of a
recombinant carboxypeptidase G2::anti-CEA scFv fusion protein",
Immunotechnology, 2:47-57. cited by applicant .
Modak et al., 2001, "Monoclonal antibody 8H9 targets a novel cell
surface antigen expressed by a wide spectrum of human solid
tumors", Cancer Res., 61:4048-54. cited by applicant .
Powers et al., 2001, "Expression of single-chain Fv-Fc fusions in
pinchia pastoris", Journal of Immunological Methods, 251:123-135.
cited by applicant .
Raag, R. and Whitlow, M., 1995, "Single-chain Fvs", FASEB Journal,
9:73-80. cited by applicant .
Schultz et al., 2000, "A tetravalent single-chain
antibody-streptavidin fusion protein for pretargeted lymphoma
therapy", Cancer Research, 60:6663-6669. cited by applicant .
Shu et al., 1993, "Secretion of a single-gene-encoded
immunoglobulin from myeloma cells", Proceedings of the National
Academy of Sciences of the United States of America, 90:7995-9.
cited by applicant .
Thanavala et al., 1986, "A surrogate hepatitis B virus antigenic
epitope represented by a synthetic peptide and an internal image
antiidiotype antibody", Journal of Experimental Medicine,
164:227-236. cited by applicant .
Towbin et al., 1979, "Electrophoretic transfer of proteins from
polyacrylamide gels to nitrocellulose sheets: procedure and some
applications", Proceedings of the National Academy of Sciences of
the United States of America, 76:4350-4. cited by applicant .
Tur et al., 2001, "Selection of scFv phages on intact cells under
low pH conditions leads to a significant loss of insert-free
phages", Biotechniques, 30:404-413. cited by applicant .
Umana et al., 1999. "Engineered glycoforms of an antineuroblastoma
IgG1 with optimized antibody-dependent cellular cytotoxic
activity", Nature Biotechnology, 17:176-180. cited by applicant
.
Wagner et al., 1997, "Immunological responses to the
tumor-associated antigen CA125 in patients with advanced ovarian
cancer induced by the murine monoclonal anti-idiotype vaccine
ACA125", Hybridoma, 16:33-40. cited by applicant .
Wang et al., 1999, "Human single-chain Fv immunoconjugates targeted
to a melanoma-associated chondroitin sulfate proteoglycan mediate
specific lysis of human melanoma cells by natural killer cells and
complement", Proceedings of the National Academy of Sciences of the
United States of America, 96:1627-32. cited by applicant .
Watters et al., 1997, "An optimized method for cell-based phage
display panning", Immunotechnology, 3:21-9. cited by applicant
.
Winter, G. and Milstein, C., 1991, "Man-made antibodies", Nature,
349:293-299. cited by applicant .
Winter et al., 1994, "Making antibodies by phage display
technology", Annual Review of Immunology, 12: 433-55. cited by
applicant .
Wright, A. and Morrison, S.L., 1997, "Effect of glycosylation on
antibody function: implications for genetic engineering", Trends in
Biotechnology, 15:26-31. cited by applicant .
Wu et al., 1996, "Tumor localization of anti-CEA single-chain Fvs:
improved targeting by non-covalent dimers", Immunotechnology,
2:21-36. cited by applicant.
|
Primary Examiner: Aeder; Sean
Attorney, Agent or Firm: Law Offices of Albert Wai-Kit Chan,
PLLC
Government Interests
The invention disclosed herein was made with government support
under Department of Energy Grant No. DE-FG-02-93ER61658
(1997-2002), the National Cancer Institute Grant No. NCI CA 89936
(Dec. 1, 2000-Nov. 30, 2002), and National Institutes of Health
Grant No. CA61017. Accordingly, the U.S. Government has certain
rights in this invention.
Parent Case Text
This application is a Divisional Application of U.S. Ser. No.
10/505,658, filed Aug. 20, 2004 now U.S. Pat. No. 7,740,845, which
is a National Stage application of PCT/US03/07004, filed Mar. 6,
2003, which is a continuation-in-part of U.S. Ser. No. 10/273,762,
filed Oct. 17, 2002 now U.S. Pat. No. 7,666,424, and
PCT/US02/33331, filed Oct. 17, 2002, which are both
continuation-in-part of U.S. Ser. No. 10/097,558, filed Mar. 8,
2002 now U.S. Pat. No. 7,737,258, which is a continuation-in-part
of PCT/US01/32565, filed Oct. 18, 2001, which claims benefit of
U.S. Ser. No. 60/241,344, filed Oct. 18, 2000, and U.S. Ser. No.
60/330,396, filed Oct. 17, 2001.
Throughout this application, various references are cited.
Disclosures of these publications in their entireties are hereby
incorporated by reference into this application to more fully
describe the state of the art to which this invention pertains.
Claims
What is claimed is:
1. A method for imaging a tumor in a subject, comprising
administering to the subject a labeled monoclonal antibody 8H9,
wherein the imaging provides information regarding the spatial
distribution of absorbed dose within a target volume.
2. The method of claim 1, wherein the antibody comprises SEQ ID
NOs: 29-34.
3. The method of claim 1, wherein the antibody is radiolabeled.
Description
BACKGROUND OF THE INVENTION
Tumor-restricted surface antigens may be targets for diagnosis and
immune-based therapies. Monoclonal antibody 8H9 is a murine IgG1
hybridoma derived from the fusion of mouse myeloma SP2/0 cells and
splenic lymphocytes from BALB/c mice immunized with human
neuroblastoma. By immunohistochemistry, 8H9 was highly reactive
with human brain tumors, childhood sarcomas, neuroblastomas and
less so with adenocarcinomas. Among primary brain tumors, 15/17
glioblastomas, 3/4 mixed gliomas, 4/11 oligodendrogliomas, 6/8
astrocytomas, 2/2 meningiomas, 3/3 schwannomas, 2/2
medulloblastomas, 1/1 neurofibroma, 1/2 neuronoglial tumors, 2/3
ependymomas and 1/1 pineoblastoma were tested positive. Among
sarcomas, 21/21 Ewing's/PNET, 28/29 rhabdomyosarcoma, 28/29
osteosarcomas, 35/37 desmoplastic small round cell tumors, 2/3
synovial sarcomas, 4/4 leiomyosarcomas, 1/1 malignant fibrous
histiocytoma and 2/2 undifferentiated sarcomas tested positive with
8H9. 87/90 neuroblastomas, 12/16 melanomas, 3/4 hepatoblastomas,
7/8 Wilm's tumors, 3/3 rhabdoid tumors and 12/27 adenocarcinomas
also tested positive. In contrast 8H9 was nonreactive with normal
human tissues including bone marrow, colon, stomach, heart, lung,
muscle, thyroid, testes, pancreas, and human brain (frontal lobe,
cerebellum, pons and spinal cord). Reactivity with normal
cynomolgus monkey tissue was similarly restricted. Indirect
immunofluorescence localized the antigen recognized by 8H9 to the
cell membrane. The antigen is proteinase-sensitive and is not
easily modulated off cell surface. 8H9 immuno-precipitated a 58 kD
band following N-glycanase treatment, most likely a protein with
heterogeneous degree of glycosylation. This novel antibody-antigen
system may have potential for tumor targeting.
Monoclonal antibodies such as 3F8 (1) and 14.18 (2) against
G.sub.D2 in neuroblastoma, M195 against CD33 in acute leukemia (3),
anti-HER2 antibodies in breast cancer (4) and anti-CD20 antibodies
in lymphoma (5) have shown efficacy in recent clinical trials. The
prognosis in glial brain tumors and metastatic mesenchymal and
neuroectodermal tumors remains dismal despite innovations in
chemotherapy and radiation therapy. Immunotherapy may offer new
possibilities for improving the outcome in these patients.
Tumor antigens expressed on cell membrane are potential targets in
immunotherapy. Examples of tumor antigens expressed on glial tumors
include neural cell adhesion molecules (6), gangliosides such as
G.sub.D2 and G.sub.M2 (7), and neurohematopoeitic antigens (8).
Recent investigations have focused on growth factor receptors as
immune targets, in particular type III mutant epidermal growth
factor receptor (EGFRvIII) which has been shown to be expressed on
50% of glial brain tumors (9). Notwithstanding the universal
expression of NCAM by neuronal cells, two clinical studies have
utilized anti-NCAM antibodies in patients. MAb UJ13A was shown to
accumulate in gliomas by virtue of disruption of blood brain bather
locally (10) and another antibody, ERIC-1 was used in a therapeutic
setting in resected glioma cavities with some clinical benefit
(11)
Recent studies have targeted immunotherapy to extracellular matrix
around tumor cells. Tenascin has been reported to be expressed in
50-95% of glial brain tumors as well as on mesenchymal tumors,
carcinomas and normal human glial, liver and kidney cells (12).
Anti-tenascin monoclonal antibodies 8106 (13) and BC-2 and BC-4
(14) administered intra-cavity have recently been reported to show
efficacy in the treatment of patients with malignant gliomas.
However, since these antigens are also present to varying degrees
on normal human neural and non-neural cells, their clinical utility
would depend on their overexpression by brain tumors when compared
to normal tissues. With the exception of EGFRvIII, the glial tumors
antigens described to date are generally found on normal brain
tissue, or are restricted to intracellular compartments, thus with
limited clinical utility for antibody targeting.
Membrane antigens that have been targeted on osteosarcoma include
G.sub.D2 (15), CD55 (16) and an as yet undefined
osteosarcoma-associated antigen recognized by the MoAbs TP-1 and
TP-3 (17). However, these antigens are present to varying degrees
on normal tissues.
Similarly the glycoprotein p30/32 coded by the MIC2 oncogene and
recognized by the monoclonal antibody O13 in the Ewing's family of
tumors is expressed on normal tissues (18). In rhabdomyosarcoma,
the MyoD family of oncofetal proteins is nuclear in localization
(19) and therefore inaccessible to antibody-targeted
immunotherapy.
An ideal tumor antigen for targeted immunotherapy should be absent
on normal tissues and abundantly expressed on tumor cell surface.
Such tumor-specific antigens e.g. idiotypes in B cell lymphoma are
rare (20). Moreover, a "generic" tumor-specific antigen expressed
on tumor cells of varying lineage recognized by monoclonal
antibodies may have broader utility in antibody-based strategies.
We describe here a novel tumor-associated antigen, recognized by a
murine monoclonal antibody 8H9, expressed on cell membranes of a
broad spectrum of tumors of neuroectodermal, mesenchymal and
epithelial origin, with restricted distribution on normal
tissues.
SUMMARY OF THE INVENTION
This invention provides a composition comprising an effective
amount of monoclonal antibody 8H9 or a derivative thereof and a
suitable carrier. This invention provides a pharmaceutical
composition comprising an effective amount of monoclonal antibody
8H9 or a derivative thereof and a pharmaceutically acceptable
carrier.
This invention also provides an antibody other than the monoclonal
antibody 8H9 comprising the complementary determining regions of
monoclonal antibody 8H9 or a derivative thereof, capable of binding
to the same antigen as the monoclonal antibody 8H9.
This invention provides a substance capable of competitively
inhibiting the binding of monoclonal antibody 8H9. In an embodiment
of the substance, it is an antibody.
This invention provides an isolated antibody, wherein the
Complementary Determining Region is NYDIN, for CDR1, WIFPGDGSTQY
for CDR2, QTTATWFAY for CDR3 for the heavy chain, and RASQSISDYLH
for the CDR1, YASQSIS for CDR2, QNGHSFPLT for CDR3 for the light
chain. This invention further provides the above antibody, wherein
the other sequences are of human origin.
The invention also provides a composition comprising an effective
amount of monoclonal antibody 8H9 or a derivative thereof and a
suitable carrier, which includes sequences as set forth in FIG. 33.
In an embodiment, the sequences are mutated. This invention also
provides the mutated form of 8H9, so as to reduce background and
cytotoxicity. Other mutations could be established which could
achieve the above-described function. In a further embodiment, the
antibody includes sequences as set forth in FIG. 34.
Furthermore, the invention provides a composition comprising the
above antibodies and an isolated nucleic acid molecule encoding the
antibodies above. This invention also provides the isolated nucleic
acid molecule above, wherein the sequences are set forth in FIG.
33.
In addition, this invention provides a vector comprising the above
nucleic acid molecules. The invention also provides a cell
comprising the above vector.
This invention provides an isolated scFv of monoclonal antibody 8H9
or a derivative thereof. In an embodiment, the scFv is directly or
indirectly coupled to a cytotoxic agent.
This invention provides a cell comprising 8H9-scFv. In an
embodiment, it is a red cell. This invention also provides a
8H9-scFv-gene modified cell. This invention provides a liposome
modified by 8H9-scFv.
This invention provides a method for directly kill, or deliver
drug, DNA, RNA or derivatives thereof to cell bearing the antigen
recognized by the monoclonal antibody 8H9 or to image cells or
tumors bearing said antigen using the isolated 8H9-scFv or cell or
liposome comprising the 8H9-scFv.
This invention provides a protein with about 58 kilodaltons in
molecular weight, reacting specifically with the monoclonal
antibody 8H9. When this 58 kd protein is glycosylated, the apparent
molecular weight is about 90 kilodaltons.
This invention also provides an antibody produced by immunizing the
8H9 antigen or specific portion thereof, which is immunogenic.
This invention also provides a nucleic acid molecule encoding the
8H9 antigen. In addition, this invention provides a nucleic acid
molecule capable of specifically hybridizing the molecule encoding
the 8H9 antigen. The nucleic acid molecule includes but is not
limited to synthetic DNA, genomic DNA, cDNA or RNA.
This invention provides a vector comprising the nucleic acid
molecule encoding 8H9 antigen or a portion thereof. This invention
provides a cell comprising the nucleic acid molecule encoding 8H9
antigen.
This invention provides a method for producing the protein which
binds to the monoclonal antibody 8H9 comprising cloning the nucleic
acid molecule encoding the 8H9 antigen in an appropriate vector,
expressing said protein in appropriate cells and recovery of said
expressed protein.
This invention also provides a method for production of antibody
using the protein produced by the above method. This invention also
provides antibodies produced by the above method. In an embodiment,
the antibody is a polyclonal antibody. In another embodiment, the
antibody is a monoclonal.
This invention provide a method of inhibiting the growth of tumor
cells comprising contacting said tumor cells with an appropriate
amount of monoclonal antibody 8H9 or a derivative thereof, or the
antibody of claim produced by the expressed 8H9 antigen or a
derivative of the produced antibody thereof.
This invention provides a method of inhibiting the growth of tumor
cells in a subject comprising administering to the subject an
appropriate amount of monoclonal antibody 8H9 or a derivative
thereof, or the antibody produced by the expressed 8H9 antigen or a
derivative thereof.
This invention provides a method for imaging a tumor in a subject
comprising administering to the subject a labeled monoclonal
antibody 8H9 or labeled derivatives, or a labeled antibody produced
by the expressed 8H9 antigen or a labeled derivative. In
embodiment, the antibodies or derivatives are labeled by a
radioisotope.
This invention provides a method of reducing tumor cells in a
subject comprising administering to the subject monoclonal antibody
8H9 or a derivative thereof, or a monoclonal antibody produced by
the expressed 8H9 antigen or a derivative thereof wherein the
antibody or derivative is coupled to a cytotoxic agent to the
subject.
This invention provides a method to evaluate the tumor bearing
potential of a subject comprising measuring the expression the 8H9
antigen in the subject, wherein the increased expression of said
antigen indicates higher tumor bearing potential of the
subject.
This invention provides a transgenic animal comprising an exogenous
gene encoding the 8H9 antigen. This invention also provides a knock
out animal wherein the gene encoding the 8H9 mouse analogous
antigen has been knocked out.
Finally, this invention provides a method to screening new
anti-tumor compound comprising contacting the above transgenic
animal with the tested compound and measuring the level of
expression of the 8H9 antigen in said transgenic animal, a decrease
in the level of expression indicating that the compound can inhibit
the expression of the 8H9 antigen and is a anti-tumor
candidate.
DETAILED DESCRIPTION OF THE FIGURES
First Series of Experiments
FIG. 1. (1A) Desmoplastic small round cell tumor (10.times.)
immunostained with 8H9 showing strong membrane positivity and
typical histology (1B) Glioblastoma multiforme stained with 8H9
showing binding to cell membranes and fibrillary stroma (1C)
Embryonal rhabdomyosarcoma stained with 8H9 showing cell membrane
reactivity (1D) Negative staining of embryonal rhabdomyosarcoma
with MOPC21, an irrelevant IgG1 control antibody
FIG. 2. Persistence of 8H9 binding to U2OS cells (2A) and NMB7
cells (2B) as studied by indirect immunofluorescence. X-axis:
relative immunofluorescence, y-axis: hours of incubation. U2OS
cells were reacted with 8H9 and HB95, and NMB7 cells with 8H9 and
3F8. After washing, cells were recultured and persistence of
immunoreactivity of the primary antibodies evaluated by indirect
immunofluorescence using FITC-conjugated secondary antibody.
Relative immunofluorescence of 8H9 on U2OS cells dropped to 80%
after 48 hrs (HB95 to 11%), while that on NMB7 cells showed no
significant drop off at 36 his (3F8 dropped to 39%)
FIG. 3. Effect of Pronase E on 8H9 immunoreactivity with HTB82,
U2OS and NMB7 cells and on 3F8 immunoreactivity with NMB7 cells as
studied by indirect immunofluorescence. X-axis: concentration of
Pronase E (mg/ml); y-axis: relative immunofluorescence
Second Series of Experiments
FIG. 4. 4 cycles of 3F8 and low level HAMA response are associated
with prolonged survival.
FIG. 5. Improved long-term survival after MoAb 3F8 in patients with
stage 4 NB newly diagnosed >1 year of age at Memorial
Sloan-Kettering Cancer Center. N4 to N7 are sequential protocols
over 15 years. N4 and N5 are chemotherapy+ABMT, N6 is
chemotherapy+3F8, and N7 is N6+.sup.131I-3F8.
FIG. 6. Antigen modulation following binding to 8H9.
FIG. 7. At 120 h: .sup.125I8H9 localized to tumors (N=4) while
control antibody 2C9 (mouse IgG1) remained in blood pool/liver
(N=4).
FIG. 8. High tumor-tissue ratio was specific for .sup.125I-8H9 vs
control MoAb .sup.125I-2C9 in RMS xenografts.
Third Series of Experiments
FIG. 9. Reactivity of 8H9 with Ewing's sarcoma cell lines.
Flow cytometric analysis of 8H9 binding to nine Ewing's sarcoma
cell lines is shown. The designation for each line is shown in the
upper right corner. FL1 fluorescence of isotype (dashed black line)
CD99 (thin black line) and 8H9 (thick black line) is shown.
FIG. 10. Lack of Reactivity of 8H9 with T cells or bone marrow
progenitor cells. Electronically gated Cd3+ cells from peripheral
blood of a normal donor (top panel) are analyzed for isotype
(dashed line), CD99 (thin black line) and 8H9 (thick black line).
Electronically gated CD34+ cells from fresh human bone marrow from
a normal donor (bottom panel) are analyzed for isotype (dashed
line) and 8H9 (thick black line) staining.
FIG. 11. Real-time PCR analysis of t(11,22) in artificially
contaminated PBMCs accurately quantifies EWS/FlI 1 transcript over
up to five log dilutions of tumor. Crossing time (x axis) is
plotted vs. fluorescence (y axis) 11a: Non-nested PCR of
10.times.10.sup.6 PBMCs contaminated from 1:10 to 1:10.sup.6. In
the inset, a linear relationship between crossing time and log cell
concentration over 4 log dilutions of tumor is shown. Samples
contaminated at less that 1:10.sup.4 show no detectable positivity
in this assay. 11b: Nested PCR of 10.times.10.sup.6 PBMCs
contaminated from 1:10 to 1:10.sup.7. A linear relationship is
observed over 5 log dilutions of tumor from 1:100 to
1:10.sup.6.
FIG. 12. Quantitative PCR analysis of purging demonstrates 2-3 log
reduction in peripheral blood and progenitor cells spikes with
Ewing's Sarcoma cells. Cycle number (x axis) is plotted vs.
fluorescence (y axis). Experimental samples were run with standard
contaminated dilutions shown in the inset 12a: Non-nested PCR
analysis of 1.times.10.sup.6 pre-purged and post-purged non-CD34
selected bone marrow from a normal healthy donor contaminated at a
level of 1:100. A two-log reduction in tumor burden is demonstrated
in the post-purged sample which shows a level of contamination at
1:10.sup.4. 12b: Nested PCR analysis of pre-purged and post-purged
CD34 selected cells harvested following G-CSF mobilization from a
patient with Ewing's sarcoma. Since this patient was negative for
EWS/FL1, CD34 cells were spike with Ewing's sarcoma at a level of
1:10.sup.3. A three-log reduction in tumor burden is demonstrated
in the post-purged sample which shows a level of contamination at
1:10.sup.6. 12c: Nested PCR analysis of pre-purged and post-purged
PBMCc from a normal healthy donor buffy coat contaminated at a
level of 1:100. A greater than 3-log reduction in tumor burden is
demonstrated in the post-purged sample which shows a level of
contamination of less than 1:10.sup.6. 12d: Nested PCR analysis of
pre-purged and post-purged non PBMCs from a normal healthy donor
buffy coat contaminated at a level of 1:10.sup.3. A 3 log reduction
in tumor burden is demonstrated in the post-purged sample which
shows a level of contamination at 1:10.sup.6.
FIG. 13. Contamination of patient elutriated apheresis fractions is
demonstrated at level of 1:10.sup.5-1:10.sup.6. Quantitative PCR
analysis of apheresis fractions from patients presenting with
disseminated Ewing's sarcoma. Cycle number (x axis) is plotted vs.
fluorescence (y axis) Patient samples are compared to standard
contaminated dilutions. Patient a (top panel) shows contamination
of all fractions at a level of 1:10.sup.5-1:10.sup.6. Patient B
(middle panel) shows contamination in the leukocyte fraction only
at a level of approximately 1:10.sup.6, Patient C (bottom panel)
shows contamination in several fractions at a similar level.
FIG. 14. Progenitor CFU capability is not affected by 8H9 based
purging. Colony forming units from CD34 selected cells from bone
marrow from a normal healthy donor (x axis) are plotted for pre-
and post purged samples.
FIG. 15. OKT3 mediated T cell proliferation is unchanged after
purging when compared to pre-purged proliferation. T cells from
normal healthy donor buffy coat were evaluated for [.sup.3H]
Thymidine uptake as a measure of T cell proliferation with a
decreasing concentration of OKT3. Uptake is measured as counts per
million (y axis) and is plotted vs. OKT3 concentration for
pre-purged (solid square), and post purged (solid circle).
Fourth Series of Experiments
FIG. 16. DSRCT (40.times.) demonstrating cell membrane and stromal
reactivity with 3F8
FIG. 17. DSRCT (40.times.) showing strong, homogeneous, cell
membrane and stromal reactivity with 8H9
Fifth Series of Experiments
FIG. 18. Inhibition of 8H9 by anti-idiotype 2E9 by FACS analysis.
18A: Staining of LAN-1 neuroblastoma cells with 5 ug/ml of 8H9
(shaded peak) was not inhibited at low concentration of 2E9 (2
ug/ml, solid line), but almost completely at concentration of 10
ug/ml (dotted line) superimposable with the negative antibody
control (solid line). 18B: Staining of LAN-1 neuroblastoma cells
with 5 ug/ml of 3F8 (anti-GD2, shaded peak) was not inhibited by
any concentrations (2 ug/ml, solid line, or 200 ug/ml, dotted line)
of 2E9. 18C Staining of HTB-82 rhabdomyosarcoma cells with 5 ug/ml
of 8H9 (grey peak) was not inhibited at low concentration (2 ug/ml,
solid line), but completely at 10 ug/ml of 2E9 (solid line)
superimposable with negative antibody control (black peak).
FIG. 19. SDS-PAGE (lanes a and b) and Western blot (c and d) of 8H9
scFv-Fc. H=heavy chain of 8H9, L=light chain of 8H9,
scFv-Fc=chimeric fusion protein between 8H9 scFv and the human
.quadrature.1-CH2-CH3 domain. With 2-mercaptoethanol: lanes a, b
and c. Native gel: lane d. SDS-PAGE was stained with Coomassie
Blue; western blot with 2E9 anti-idiotypic antibody.
FIG. 20. FACS analysis of 8h9-scFv-Fc staining of HTB82
rhabdomyosarcoma cells. 20A Immunofluorescence increased with
concentrations of 8H9-scFv-Fc (1, 5, 25, 125 ug/ml), shaded peak is
no antibody control. 20B: Cell staining (5 ug/ml of 8H9-scFv-Fc,
thin solid line) was completely inhibited (thick solid line) at 1
ug/ml of anti-idiotypic antibody 2E9, shaded peak is no antibody
control.
FIG. 21. Immunoscintigraphy of human tumors using .sup.125I-labeled
8H9 scFv-Fc. Mice xenografted with human LAN-1 neuroblastoma
received retroorbital injections of 25 uCi of 125I-labeled
antibody. 24 h, 48 h and 7 days after injection, the animals were
anesthetized and imaged with a gamma camera.
FIG. 22. Blood clearance of .sup.125I-labeled chimeric 8H9 and
.sup.125I-native 8H9. Mice xenografted with human LAN-1
neuroblastoma received retroorbital injections of .sup.125I-labeled
antibody. Percent injected dose/gm of serial blood samples were
plotted over time.
Sixth Series of Experiments
FIG. 23. Anti-idiotype affinity enrichment of producer lines.
Producer lines were stained with anti-idiotypic MoAb 2E9 before
(shaded peak, A and B), and after first (dotted line peak, A) and
second (thick solid line, A) affinity purification, and after first
(dotted line, B) and second (solid line B) subcloning, showing
improved scFv expression. By FACS the indicator line K562 showed
improved scFv expression after first (dotted line, C) and second
(thick solid line, C) affinity purification of the producer line,
and subsequent first (dotted line, C) and second (thick solid line,
D) subcloning of the producer line, when compared to unpurified
producer lines (shaded peaks, C and D), consistent with improvement
in gene transduction efficiency. The thin solid line curves in each
figure represents nonproducer line (A and B) or uninfected K562 (C
and D).
FIG. 24. Flow cytometry analysis of scFv expression. Cultured
8H9-scFv-CD28-. gene-modified lymphocytes were assayed for their
scFv expression using anti-idiotypic MoAb 2E9 (solid curves) and
control rat IgG1 MoAb as control (shaded histograms) from day 13
through day 62. Although a substantial proportion of cells were
positive by day 13, they became homogeneous by day 21 and persisted
till day 62, when the overall mean fluorescence appeared to
decrease.
FIG. 25. In vitro expansion of 8H9-scFv-CD28-.quadrature.
gene-modified primary human lymphocytes. Clonal expansion was
expressed as a fraction of the initial viable cell number. IL-2
(100 U/ml) was added after retroviral infection and was present
throughout the entire in vitro culture period. Short bars depict
the days when soluble anti-idiotypic antibody 2E9 (3-10 ug/ml) was
present in the culture.
FIG. 26. Cytotoxicity against tumor cell lines:
8H9-scFv-CD28-.quadrature. gene-modified lymphocytes from day 56 of
culture (scFv-T) were assayed by .sup.51Cr release assay in the
presence or absence of 8H9 (50 ug/ml final concentration) as an
antigen blocking agent. Control lymphocytes (LAK) from the same
donor but not gene-modified, were cultured under the same
conditions as the gene-modified cells. 26A: NMB-7 neuroblastoma.
26B: LAN-1 neuroblastoma. 26C: HTB-82 rhabdomyosarcoma. 26D: Daudi
lymphoma.
FIG. 27. Suppression of rhabdomyosarcoma tumor growth in SCID mice.
Human rhabdomyosarcoma HTB-82 was strongly reactive with 8H9, but
not with 5F11 (anti-GD2) antibodies. Experimental group:
8H9-scFv-CD28-.quadrature. gene-modified human lymphocytes (solid
circles). Control groups: no cells+2E9 (open circles), cultured
unmodified lymphocytes (LAK)+2E9 (open triangles), or
5F11scFv-CD28-.quadrature. modified lymphocytes+1G8 [rat anti-5F11
anti-idiotype MoAb] (solid triangles).
Seventh Series of Experiments
FIG. 28. Sequential imaging of nude mouse bearing RMS xenograft 24,
48 and 172 h after injection with 4.4 MBq .sup.125I-8H9 as compared
to a RMS xenograft-bearing mouse imaged 172 h after injection with
4.4 MBq .sup.125I-2C9.
FIG. 29. Blood kinetics of .sup.125I-8H9 in nude mice with RMS
xenografts. Error bars represent SEM.
FIG. 30. Comparison of biodistribution of .sup.125I-8H9 at 24, 48
and 172 h after injection in xenograft and normal tissues.
FIG. 31. Comparison of biodistribution of .sup.125I-8H9 with that
of the nonspecific anticytokeratin MoAb .sup.125I-2C9 (solid bars)
in xenografts and normal tissues.
FIG. 32. Anti tumor effect on RMS xenografts: .sup.131I-8H9 versus
negative control MoAb .sup.131I-3F8. Each mouse received 18.5 MBq
radiolabeled MoAb (5 mice per group).
FIG. 33: 8H9 scFv gene sequence (sense and complementary).
Complementary determining regions (CDR) are marked in boxes in the
following order: CDR-1 (HC, heavy chain), CDR-2 (HC), CDr-3 (HC),
CDR-1 (LC, light chain), CDR-2 (LC), CDR-3 (LC).
FIG. 34: Gene and amino acid sequences of 8H9scFv is depicted.
Mutated 8H9 scFv carries the following site-directed mutagenesis
(VH: K13E and VL: R18Q, R45Q, K103E, K107E) to decrease PI from 6.4
to 4.8, and net charge from -1 to -9, a strategy to decrease
nonspecific normal tissue adherence.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a composition comprising an effective
amount of monoclonal antibody 8H9 or a derivative thereof and a
suitable carrier. This invention provides a pharmaceutical
composition comprising an effective amount of monoclonal antibody
8H9 or a derivative thereof and a pharmaceutically acceptable
carrier. In an embodiment of the above composition, the derivative
is a scFv. In a separate embodiment, the antibody is an
antibody-fusion construct. In another embodiment, the antibody is
an scFvFc.
This invention provides an isolated antibody, wherein the
Complementary Determining Region is NYDIN (SEQ ID NO:29) for CDR1,
WIFPGDGSTQY (SEQ ID NO:30) for CDR2, QTTATWFAY (SEQ ID NO:31) for
CDR3 for the heavy chain, and RASQSISDYLH (SEQ ID NO:32) for the
CDR1, YASQSIS (SEQ ID NO:33) for CDR2, QNGHSFPLT (SEQ ID NO:34) for
CDR3 for the light chain. This invention further provides the above
antibody, wherein the other sequences are of human origin.
The invention also provides a composition comprising an effective
amount of monoclonal antibody 8H9 or a derivative thereof and a
suitable carrier, which includes sequences as set forth in FIG. 33.
In an embodiment, the sequences are mutated. This invention also
provides the mutated form of 8H9, so as to reduce background and
cytotoxicity. Other mutations could be established which could
achieve the above-described function. In a further embodiment, the
antibody includes sequences as set forth in FIG. 34.
Furthermore, the invention provides a composition comprising the
above antibodies and an isolated nucleic acid molecule encoding the
antibodies above. This invention also provides the isolated nucleic
acid molecule above, wherein the sequences are set forth in FIG.
33.
This invention also provides a peptide or polypeptide comprising:
NYDIN (SEQ ID NO:29), WIFPGDGSTQY (SEQ ID NO:30), QTTATWFAY (SEQ ID
NO:31), RASQSISDYLH (SEQ ID NO:32), YASQSIS (SEQ ID NO:33),
QNGHSFPLT (SEQ ID NO:34) alone or in combination thereof. The
peptide or polypeptide functions similarly to the 8H9 antibody and
therefore, for the below uses, this peptide or polypeptide may be
used similarly. In an embodiment, the invention provides an
isolated nucleic acid encoding the above peptide.
In addition, this invention provides a vector comprising the above
nucleic acid molecules. The invention also provides a cell
comprising the above vector. This invention further provides cells
expressing the 8H9 or derivative of 8H9 antibodies.
This invention provides an antibody other than the monoclonal
antibody 8H9 comprising the complementary determining regions of
monoclonal antibody 8H9 or a derivative thereof, capable of binding
to the same antigen as the monoclonal antibody 8H9.
This invention also provides a substance capable of competitively
inhibiting the binding of monoclonal antibody 8H9. In an
embodiment, the substance is an antibody. In another embodiment,
the substance is a peptide.
This invention further provides an isolated single chain antibody
of 8H9 or a derivative thereof. This invention also provides an
isolated scFv of monoclonal antibody 8H9 or a derivative thereof.
Single chain antibodies or derivatives are known in the antibody
field. In an embodiment, the scFv is directly or indirectly coupled
to a cytotoxic agent. In a further embodiment, the scFv is linked
to a first protein capable of binding to a second protein which is
coupled to a cytotoxic agent. Same rationale applies to the imaging
uses of the 8H9 monoclonal antibody or its derivative. In the case
of imaging, instead of a cytotoxic agent, the antibody or its
derivative will be coupled to an imaging agent. Both cytotoxic or
imaging agents are known in the art.
This invention provides a cell comprising 8H9-scFv. In an
embodiment, the cell is a red cell. This invention also provides a
8H9-scFv-gene modified cell.
This invention also provides a liposome modified by 8H9-scFv. It is
one intention of the disclosure to deliver 8H9 via liposomes or via
other delivery technologies.
This invention provides a method to directly kill, or deliver drug,
DNA, RNA or derivatives thereof to cell bearing the antigen
recognized by the monoclonal antibody 8H9 or to image cells or
tumors bearing said antigen using the isolated 8H9-scFv or 8H9-scFv
modified cell or liposome.
This invention provides a protein with about 58 kilodaltons in
molecular weight, reacting specifically with the monoclonal
antibody 8H9. When this protein is glycosylated, the apparent
molecular weight is about 90 kilodaltons.
This invention provides an antibody produced by immunizing the
expressed 8H9 antigen or specific portion thereof. The specific
portion is the region which is recognized by 8H9. Another region is
the region which performs its specific function.
This invention provides a nucleic acid molecule encoding 8H9
antigen.
This invention provides a nucleic acid molecule capable of
specifically hybridizing the nucleic acid molecule which encodes
the 8H9 antigen. The nucleic acid molecule includes but is not
limited to synthetic DNA, genomic DNA, cDNA or RNA.
This invention also provides a vector comprising the nucleic acid
molecule encoding the 8H9 antigen or a portion thereof. The portion
could be a functional domain of said antigen. This invention
provides a cell comprising the nucleic acid molecule encoding the
8H9 antigen.
This invention provides a method for producing the protein which
binds to the monoclonal antibody 8H9 comprising cloning the nucleic
acid molecule which encodes the 8H9 antigen in an appropriate
vector, expressing said protein in appropriate cells and recovery
of said expressed protein.
This invention provides a method for production of antibody using
the expressed 8H9 antigen or the portion which is immunogenic. This
invention also provides an antibody produced by the above described
method. In an embodiment, the antibody is polyclonal. In another
embodiment, the antibody is a monoclonal.
This invention provides a method of inhibiting the growth of tumor
cells comprising contacting said tumor cells with an appropriate
amount of monoclonal antibody 8H9 or a derivative thereof, or the
antibody produced using the expressed 8H9 antigen or a derivative
thereof.
This invention provides a method of inhibiting the growth of tumor
cells in a subject comprising administering to the subject an
appropriate amount of monoclonal antibody 8H9 or a derivative
thereof, or the antibody produced using the expressed 8H9 antigen
or a derivative thereof.
This invention provides a method for imaging a tumor in a subject
comprising administering to the subject a labeled monoclonal
antibody 8H9 or a labeled derivatives, or a labeled antibody
produced using the expressed 8H9 antigen or a labeled derivative.
In an embodiment, the antibody or the derivative is labeled with
radioisotope.
This invention provides a method of reducing tumor cells in a
subject comprising administering to the subject monoclonal antibody
8H9 or a derivative thereof, or a monoclonal antibody produced
using the expressed 8H9 antigen or a derivative thereof wherein the
antibody or derivative is coupled to a cytotoxic agent to the
subject. In an embodiment, the coupling to a cytotoxic agent is
indirect. In another embodiment, the coupling is first directly
linking the antibody or derivative with a first protein which is
capable of bind to a second protein and the second protein is
covalently couple to a cytotoxic agent. In a further embodiment,
the cytotoxic agent is a radioisotope.
This invention also provides a method to evaluate the tumor bearing
potential of a subject comprising measuring the expression the 8H9
antigen in the subject, wherein the increased expression of said
antigen indicates higher tumor bearing potential of the
subject.
This invention provides a transgenic animal comprising an exogenous
gene encoding the 8H9 antigen. The transgenic animal may also
carried an knock out gene encoding the 8H9 mouse analogous antigen.
In an embodiment, it is a transgenic mouse.
This invention provides a method to screening new anti-tumor
compound comprising contacting the transgenic animal with the
tested compound and measuring the level of expression of the 8H9
antigen in said transgenic animal, a decrease in the level of
expression indicating that the compound can inhibit the expression
of the 8H9 antigen and is a anti-tumor candidate.
The invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative, and are not meant to limit the invention as
described herein, which is defined by the claims which follow
thereafter.
EXPERIMENTAL DETAILS
First Series of Experiments
Monoclonal Antibody 8H9 Targets a Novel Cell Surface Antigen
Expressed by a Wide Spectrum of Human Solid Tumors
Tumor-restricted surface antigens may be targets for diagnosis and
immune-based therapies. Monoclonal antibody 8H9 is a murine IgG1
hybridoma derived from the fusion of mouse myeloma SP2/0 cells and
splenic lymphocytes from BALB/c mice immunized with human
neuroblastoma. By immunohistochemistry, 8H9 was highly reactive
with human brain tumors, childhood sarcomas, neuroblastomas and
less so with adenocarcinomas. Among primary brain tumors, 15/17
glioblastomas, 3/4 mixed gliomas, 4/11 oligodendrogliomas, 6/8
astrocytomas, 2/2 meningiomas, 3/3 schwannomas, 2/2
medulloblastomas, 1/1 neurofibroma, 1/2 neuronoglial tumors, 2/3
ependymomas and 1/1 pineoblastoma were tested positive. Among
sarcomas, 21/21 Ewing's/PNET, 28/29 rhabdomyosarcoma, 28/29
osteosarcomas, 35/37 desmoplastic small round cell tumors, 2/3
synovial sarcomas, 4/4 leiomyosarcomas, 1/1 malignant fibrous
histiocytoma and 2/2 undifferentiated sarcomas tested positive with
8H9. 87/90 neuroblastomas, 12/16 melanomas, 3/4 hepatoblastomas,
7/8 Wilm's tumors, 3/3 rhabdoid tumors and 12/27 adenocarcinomas
also tested positive. In contrast 8H9 was nonreactive with normal
human tissues including bone marrow, colon, stomach, heart, lung,
muscle, thyroid, testes, pancreas, and human brain (frontal lobe,
cerebellum, pons and spinal cord). Reactivity with normal
cynomolgus monkey tissue was similarly restricted. Indirect
immunofluorescence localized the antigen recognized by 8H9 to the
cell membrane. The antigen is proteinase-sensitive and is not
easily modulated off cell surface. 8H9 immuno-precipitated a 58 kD
band following N-glycanase treatment, most likely a protein with
heterogeneous degree of glycosylation. This novel antibody-antigen
system may have potential for tumor targeting.
Monoclonal antibodies such as 3F8 (1) and 14.18 (2) against
G.sub.D2 in neuroblastoma, M195 against CD33 in acute leukemia (3),
anti-HER2 antibodies in breast cancer (4) and anti-CD20 antibodies
in lymphoma (5) have shown efficacy in recent clinical trials. The
prognosis in glial brain tumors and metastatic mesenchymal and
neuroectodermal tumors remains dismal despite innovations in
chemotherapy and radiation therapy. Immunotherapy may offer new
possibilities for improving the outcome in these patients.
Tumor antigens expressed on cell membrane are potential targets in
immunotherapy. Examples of tumor antigens expressed on glial tumors
include neural cell adhesion molecules (6), gangliosides such as
G.sub.D2 and G.sub.M2 (7), and neurohematopoeitic antigens (8).
Recent investigations have focused on growth factor receptors as
immune targets, in particular type III mutant epidermal growth
factor receptor (EGFRvIII) which has been shown to be expressed on
50% of glial brain tumors (9). Notwithstanding the universal
expression of NCAM by neuronal cells, two clinical studies have
utilized anti-NCAM antibodies in patients. MAb UJ13A was shown to
accumulate in gliomas by virtue of disruption of blood brain bather
locally (10) and another antibody, ERIC-1 was used in a therapeutic
setting in resected glioma cavities with some clinical benefit
(11)
Recent studies have targeted immunotherapy to extracellular matrix
around tumor cells. Tenascin has been reported to be expressed in
50-95% of glial brain tumors as well as on mesenchymal tumors,
carcinomas and normal human glial, liver and kidney cells (12).
Anti-tenascin monoclonal antibodies 8106 (13) and BC-2 and BC-4
(14) administered intra-cavity have recently been reported to show
efficacy in the treatment of patients with malignant gliomas.
However, since these antigens are also present to varying degrees
on normal human neural and non-neural cells, their clinical utility
would depend on their overexpression by brain tumors when compared
to normal tissues. With the exception of EGFRvIII, the glial tumors
antigens described to date are generally found on normal brain
tissue, or are restricted to intracellular compartments, thus with
limited clinical utility for antibody targeting.
Membrane antigens that have been targeted on osteosarcoma include
G.sub.D2 (15), CD55 (16) and an as yet undefined
osteosarcoma-associated antigen recognized by the MoAbs TP-1 and
TP-3 (17). However, these antigens are present to varying degrees
on normal tissues. Similarly the glycoprotein p30/32 coded by the
MIC2 oncogene and recognized by the monoclonal antibody O13 in the
Ewing's family of tumors is expressed on normal tissues (18). In
rhabdomyosarcoma, the MyoD family of oncofetal proteins is nuclear
in localization (19) and therefore inaccessible to
antibody-targeted immunotherapy.
An ideal tumor antigen for targeted immunotherapy should be absent
on normal tissues and abundantly expressed on tumor cell surface.
Such tumor-specific antigens e.g. idiotypes in B cell lymphoma are
rare (20). Moreover, a "generic" tumor-specific antigen expressed
on tumor cells of varying lineage recognized by monoclonal
antibodies may have broader utility in antibody-based strategies.
We describe here a novel tumor-associated antigen, recognized by a
murine monoclonal antibody 8H9, expressed on cell membranes of a
broad spectrum of tumors of neuroectodermal, mesenchymal and
epithelial origin, with restricted distribution on normal
tissues.
Materials and Methods
Tumor and Normal Tissue Samples
Frozen tumors from 330 patients with neuroectodermal, mesenchymal
and epithelial neoplasia were analyzed. All diagnoses of tumor
samples were confirmed by hematoxylin and eosin assessment of
paraffin-embedded specimens. 15 normal human tissue samples and 8
normal cynomolgus monkey tissue samples obtained at autopsy were
also analyzed.
Cell Lines
Human neuroblastoma cell lines LA-N-1 was provided by Dr. Robert
Seeger, Children's Hospital of Los Angeles, Los Angeles, Calif.
Human neuroblastoma cell lines LA-15-N, LA-66-N, LA-5S, LA-19-S and
LA-19-N were provided by Dr. Robert Ross (Fordham University, NY)
and IMR 32 and NMB7 by Dr. Shuen-Kuei Liao (McMaster University,
Ontario, Canada). Breast carcinoma cell lines SW480 and ZR75-1 were
provided by Dr. S. Welt (Memorial Sloan-Kettering Cancer Center,
NY) and the melanoma line SKMe128 by Dr. P. Chapman (Memorial
Sloan-Kettering Cancer Center, NY). Neuroblastoma cell lines SKNHM,
SKNHB, SKNJD, SKNLP, SKNER, SKNMM, SKNCH and SKNSH,
rhabdomyosarcoma cell line SKRJC and Ewing's/PNET cell lines SKPPR,
SKPRT and SKNMC were derived from patients with metastatic disease
treated at MSKCC. The following cell lines were purchased from
American Type Culture Collection, Bethesda, Md.: melanoma cell
lines HTB63 and HTB67, rhabdomyosarcoma cell line HTB82, small cell
lung cancer cell line HTB 119, acute T-leukemia cell line Jurkat,
glioblastoma multiforme cell line Glio72, breast cancer cell line
HTB 22, colon carcinoma cells line SK Co-1, Hela, embryonal kidney
293, and osteosarcoma cell lines CRL1427, HTB86 and HTB 96. All
cell lines were grown at 37.degree. C. in a 6% CO.sub.2 incubator
using standard culture medium, which consisted of RPMI 1640 medium
supplemented with 10% bovine calf serum, 2 mM glutamine, penicillin
(100 IU/ml) and streptomycin (100 .mu.g/ml). Normal human
hepatocytes were purchased from Clonetics, San Diego, Calif. and
processed immediately upon delivery. Normal human mononuclear cells
were prepared from heparinized bone marrow samples by
centrifugation across a Ficoll-Hypaque density separation gradient.
EBV lymphoblastoid cell lines were derived from human mononuclear
cells.
Monoclonal Antibody
Female BALB/c mice were hyperimmunized with human neuroblastoma
according to previously outlined methods (21). Lymphocytes derived
from these mice were fused with SP2/0 mouse myeloma cells line.
Clones were selected for specific binding on ELISA. The 8H9
hybridoma secreting an IgG.sub.1 monoclonal antibody was selected
for further characterization after subcloning.
Immunohistochemical Studies
Eight .mu.m cryostat frozen tumor sections were fixed in acetone
and washed in PBS. Immunohistochemical studies were performed as
described previously (22). Endogenous peroxidases were blocked in
0.3% H.sub.2O.sub.2 in PBS. Sections were incubated in 10% horse
serum (Gibco BRL, Gaithersburg, Md.) after blocking with avidin and
biotin. Incubation with purified 8H9 (2 .mu.g/ml) in PBS was
carried out at room temperature for 1 hour. An IgG1 myeloma was
used as a control (Sigma Chemical, St Louis Mo.). Sections were
incubated with a secondary horse anti-mouse biotinylated antibody
(Vector Laboratories, Burlingame, Calif.) followed by incubation
with ABC complex (Vector) and developed with Vector VIP peroxidase
substrate or DAB peroxidase substrate kit (Vector). A 10%
hematoxylin counterstain for 4 minutes was used. Staining was
graded as positive or negative and homogeneous or heterogeneous
reactivity noted.
Indirect Immunofluorescence
1 million target cells were washed in PBS and then spun at
180.times.g for 5 min. The pellets were then reacted with 100 .mu.l
of 15 .mu.g/ml 8H9 at 4.degree. C. for 1 hour. After washing the
cells with PBS they were allowed to react with 100 .mu.l
FITC-conjugated goat F (ab').sub.2 anti-mouse IgG+IgM, (Biosource
International, Camarillo, Calif.) at 4.degree. C. Flow cytometric
analysis was performed using FACSCalibur Immunocytometer
(Becton-Dickinson Immunocytometry Systems, San Jose, Calif.).
In order to study loss of antigen after binding to 8H9, 10.sup.6
NMB7 and U2OS cell pellets were prepared as above and reacted with
100 .mu.l each of 15 .mu.g/ml of 8H9 or the anti-HLA A, B, C
antibody, HB-95 (American Type Culture Collection, Bethesda, Md.)
at 4.degree. C. for 1 hour. NMB7 cells were also similarly reacted
with the anti-G.sub.D2 monoclonal antibody 3F8. After washing with
PBS, cells were cultured at 37.degree. C. in standard culture
medium for 0, 1, 2, 4, 8, 12, 24, 36 and 48 h. They were then
reacted with FITC-conjugated secondary antibody goat F (ab').sub.2
anti-mouse IgG+IgM, (Biosource International, Camarillo, Calif.) at
4.degree. C. Flow cytometric analysis was performed. Geometric mean
immunofluorescence was compared to that of control cells incubated
for similar time intervals in standard culture medium in the
absence of MoAbs, and then immunostained with HB-95 (U2OS) or 3F8
(NMB7).
Antigen sensitivity to proteinase was tested by incubating
0.5.times.10.sup.6 of HTB82, U2OS and NMB7 cells at 37.degree. C.
for 30 minutes in RPMI with increasing concentrations of neutral
proteinase, Pronase E from streptomyces griseus (E. Merck,
Darmstadt, Germany) After washing, cells were stained with 8H9 or
3F8 and studied by indirect immunofluorescence.
Immunoprecipitation
Immunoprecipitation was carried out using a modification of the
standard technique. (23) 8H9-positive cell lines (NMB7, LAN-1,
HTB82, U2OS, HELA, 293) and 8H9-negative cell lines (Jurkat,
HTB119) were used. 2.times.10.sup.7 viable cells were washed in TBS
(0.05 M Tris-HCl, pH 8, with 0.15 M NaCl) and incubated with 10 U
lactoperoxidase (Sigma) 100 ul of 100 U/ml in TBS, 1 mCi .sup.125I
(2.7 ul) and 1/6000 dilution of 30% hydrogen peroxide for 5 min at
20.degree. C. Five units of lactoperoxidase (50 ul) and the same
dilution of hydrogen peroxide (50 ul) were added every 3 min with
mixing for a total of 3 times. The cells were washed extensively in
TBS containing 2 mg/ml of NaI. The iodinated cells were washed
three times in TBS, lysed on ice (30 min) in 500 ul of modified
RIPA buffer (0.01 M Tris-HCl, pH 7.2, 0.15 M NaCl, 1% sodium
deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS),
0.01 M EDTA) containing protease inhibitors (1 mM PMSF, 50 ug/ml
Bestatin, 2 ug/ml Aprotinin, 0.5 ug/ml Leupeptin, 0.7 ug/ml
Pepstatin, 10 ug/ml E-64). The lysates were clarified by
centrifugation at 15,000 rpm for 5 min at 4.degree. C., then
incubated with 1 mg of 8H9 or IgG1 control antibody for 16 hr at
4.degree. C. with mixing. The antigen-antibody complex was
collected by adsorption onto 100 ul Protein G-sepharose beads
(Sigma) for 6 hr at 4.degree. C. The immune complex immobilized on
Protein G was washed three times with modified RIPA buffer, and
then washed once with RIPA buffer containing 1 M NaCl, and then
twice with modified TNN buffer (0.05 M Tris-HCl, pH 8, 0.15 M NaCl,
0.05% Nonidet P-40). Bound proteins were removed by elution with
SDS-sample buffer and analyzed by 7.5% SDS-PAGE, followed by
autoradiography. Deglycosylation of the radiolabeled antigen was
carried out on the protein G sepharose using N-glycanase (Glyco,
Novato, Calif.) and O-glycanase (Glyco) according to manufacturers'
instructions. Molecular weight was estimated using Quantity One
software from BioRad Inc. (Hercules, Calif.).
Results
Immunohistochemical Studies
Frozen sections from 330 tumors with histologically confirmed
diagnoses of cancer were analyzed for immunoreactivity with mAb 8H9
(Tables 1, 2). 15 histologically normal human tissues and 8 normal
monkey tissues were also analyzed (Table 3).
TABLE-US-00001 TABLE 1 8H9 reactivity: neuroectodermal Tumors
Tumors No. 8H9 positive % Neuroblastoma 90 87 97 Brain Tumors 1.
Glial Tumors Glioblastoma multiforme 17 15 88 Mixed Glioma 4 3 --
Oligodendroglioma 11 4 36 Astrocytoma 8 6 75 Ependymoma 3 2 -- 2.
Primitive PNET Medulloblastoma 2 2 -- 3. Mixed Neuronoglial tumor 2
1 -- 4. Other Schwannoma 3 3 -- Meningioma 2 2 -- Neurofibroma 1 1
-- Pineoblastoma 1 1 -- Melanoma 16 12 75 Ewing's Family of tumors
21 21 100 TOTAL 181 160 88
TABLE-US-00002 TABLE 2 8H9 reactivity: mesenchymal, epithelial and
other tumors Tumors No. 8H9 Reactive % A. Mesenchymal
Rhabdomyosarcoma 29 28 97 Osteosarcoma 29 28 97 Desmoplastic small
round cell tumor 37 35 95 Leiomyosarcoma 4 4 -- Synovial sarcoma 3
2 -- Malignant fibrous histiocytoma 1 1 -- Undifferentiated sarcoma
2 2 -- Fibrosarcoma 1 0 TOTAL 106 100 94 B. Epithelial Breast 12 4
33 Bladder 4 1 -- Adrenal 3 1 -- Stomach 1 1 -- Prostate 2 1 --
Colon 1 1 -- Lung 1 1 -- Endometrium 1 1 -- Cervix 1 0 -- Renal 1 1
-- TOTAL 27 12 44 Epithelial tumors summary No. Slide Date
Diagnosis 8H9 1 7251 Mar. 11, 1998 Breast Ca neg 2 7279 Mar. 13,
1998 Breast Ca neg 3 7282/7601 Mar. 13, 1998; Oct. 5, Breast Ca neg
1998 4 7722 Oct. 21, 1998 Breast Ca NE (no cells) 5 7261 Mar. 11,
1998 Breast Ca pos 6 6388 Aug. 26, 1998 Breast Ca pos 7 6493 Oct.
11, 1998 Breast Ca neg 8 6498 Oct. 11, 1998 Breast Ca neg 9 6499
Oct. 11, 1998 Breast Ca neg 10 6492 Oct. 11, 1998 Breast Ca neg 11
6376 Aug. 26, 1996 Breast Ca pos 12 6488 Oct. 11, 1998 Bladder Ca
neg 13 6489 Oct. 11, 1998 Bladder Ca weakly + 14 6490 Oct. 11, 1998
Bladder Ca neg 15 6491 Oct. 11, 1998 Bladder Ca neg 16 6441 Sep.
30, 1998 Lung Ca pos 17 6503 Oct. 11, 1998 Prostate Ca neg 18 6504
Oct. 11, 1998 Prostate Ca pos 19 6501 Oct. 11, 1998 Cervix Ca neg
20 6502 Oct. 11, 1998 Uterine Ca pos 21 7717 Oct. 21, 1998 Adrenal
Ca ne (necrotic) 22 7250 Mar. 11, 1998 Adrenal Ca neg 23 7207 Nov.
18, 1997 Renal Ca pos 24 6505 Oct. 11, 1998 Stomach Ca pos 25 7886
Feb. 22, 1999 Adrenal Ca pos Total Evaluable 8H9 pos. Breast 11 10
3 of 10 Bladder 4 4 1 of 4 Prostate 2 2 1 of 2 Adrenal 3 2 1of 2
Renal 1 1 1 of 1 Stomach 1 1 1 of 1 Uterine 1 1 1 of 1 Cervix 1 1 0
of 1 Lung 1 1 1 of 1 TOTAL 25 23 10 of 23 C. Other tumors Tumors
No. 8H9 reactive % Hepatoblastoma 4 3 -- Wilm's tumor 8 7 --
Rhabdoid tumor 3 3 -- Paraganglioma 1 1 -- TOTAL 16 14 88
TABLE-US-00003 TABLE 3 8H9 reactivity in normal human and
cynomolgus monkey tissues Tissues 8H9 reactivity A. Human Frontal
lobe Negative Pons Negative Spinal cord Negative Cerebellum
Negative Lung Negative Heart Negative Skeletal muscle Negative
Thyroid Negative Testes Negative Pancreas cytoplasmic staining*
Adrenal cortex cytoplasmic staining* Liver cytoplasmic staining*
Sigmoid colon Negative Bone Marrow Negative Kidney Negative B.
Cynomolgus monkey Cerebellum Negative Frontal Lobe Negative
Occipital Cortex Negative Brainstem Negative Liver cytoplasmic
staining Stomach Negative Adrenal Cortex cytoplasmic staining
Kidney Negative *non-specific background
Heterogenous, non-specific cytoplasmic staining was noticed in
normal human pancreas, stomach, liver and adrenal cortex which was
diminished when 8H.sub.9F(ab').sub.2 fragments were used instead of
the whole antibody for immunostaining (data not shown). None of the
other human tissues showed reactivity with 8H9. In particular
normal human brain tissue sections including frontal lobe, spinal
cord, pons and cerebellum were completely negative. Normal tissues
from cynomolgus monkey also demonstrated similarly restricted
reactivity with nonspecific staining observed in stomach and liver.
(Table 3)
The majority of neuroectodermal and mesenchymal tumors tested
showed positive reactivity with 8H9, epithelial tumors to a lesser
extent. 8H9 immunoreactivity was seen in a characteristic,
homogeneous, cell membrane distribution in 286 of the 330 (87%)
tumor samples examined. (FIG. 1). 88% of neuroectodermal tumors,
94% of mesenchymal tumors and 44% of epithelial tumors tested
positive with 8H9. (Tables 2, 3)
Indirect Immunofluorescence
8H9 immunoreactivity in 35 neuroblastoma, melanoma,
rhabdomyosarcoma, small cell lung cancer, osteosarcoma,
glioblastoma, leukemia, breast cancer and colon cancer cell lines
was tested using indirect immunofluorescence. Moderate to strong
cell membrane reactivity with 8H9 was detected in 16/16
neuroblastoma cell lines, 3/3 melanoma cell lines, 2/2
rhabdomyosarcoma cell lines, 1/1 glioblastoma multiforme cell line,
3/3 breast cancer cell lines and 1/1 colon cancer cell lines
studied. 2 of 3 Ewing's/PNET cell lines, and 2 of the 3
osteosarcoma cell lines were strongly positive while the others
showed weak positivity. The small cell lung cancer cell line tested
negative with 8H9 as did Jurkat T-ALL cell line and EBV transformed
lymphoblastoid cells. Normal human bone marrow mononuclear cells
and hepatocytes had no reactivity with 8H9. (Table 4) In the
neuroblastoma cell lines studied, indirect immunofluorescence with
8H9 was weaker (mean fluorescence: 73.73; negative control: 3.95)
when compared to the anti-G.sub.D2 antibody 3F8 (mean fluorescence:
249.95).
8H9 binding to U2OS as detected by indirect immunofluorescence did
not diminish significantly after 48 hr of incubation at 37.degree.
C. During the same period, binding to the anti-HLA antibody HB-95
diminished by 89%. Similarly there was no significant loss of 8H9
binding to NMB7 cells. whereas 3F8 binding diminished by 61%. (FIG.
2)
There was a pronase dose-dependent reduction in reactivity with 8H9
with 75-85% loss of immunofluorescence achieved at a final Pronase
concentration of 0.3 mg/ml (FIG. 3). There was no appreciable loss
of reactivity with 3F8 on NMB7 cells. Furthermore, the 8H9 antigen
was not sensitive to neuraminidase or phosphatidyl-inositol
specific phospholipase C (data not shown).
TABLE-US-00004 TABLE 4 8H9 reactivity with cell lines by indirect
immunofluorescence Cell line 8H9 Reactivity 1. Neuroblastoma LA-N-1
positive NMB7 positive LA-1-15-N positive LA-1-66N positive IMR32
positive LA-1-19N positive LA-1-5S positive LA-1-19S positive SKNHM
positive SKNSH positive SKNHB positive SKNJD positive SKNLP
positive SKNMM positive SKPCH positive SKNER positive 2. Melanoma
HTB63 positive HTB67 positive SKMe128 positive 3. Rhabdomyosarcoma
HTB 82 positive SKRJC positive 4. Small cell lung cancer HTB119
negative 5. Osteosarcoma CRL1427 positive HTB96 positive HTB86
positive 6. Ewing's/PNET SKPPR positive SKPRT positive SKNMC
positive 7. Glioblastoma Glio72 positive 8. Carcinoma Breast ZR75-1
positive SW480 positive HTB22 positive 9. Carcinoma Colon SKCo-1
positive 10. Leukemia Jurkat negative 11. Normal human cells
negative Bone marrow negative Hepatocytes negative 12. EBV
lymphoblastoid cells negative
Immunoprecipitation:
8H9 immunoprecipitated a broad band centered around 90 kD from all
the 8H9-positive cell lines (HTB82, NMB7, LAN1, U2OS, Hela, 293),
whether using native or reducing (2ME) conditions (data not shown).
Neither control IgG1 antibody nor 8H9-negative cell lines (Jurkat
or HTB119) showed the 90 kD antigen. Following N-glycanase
treatment, a single 58 kD band was found. O-glycanase had no
effect. We interpreted this to mean a protein with heterogeneous
glycosylation pattern, without disulfide-linked subunits.
Discussion
We describe a novel 58 kD surface tumor antigen, which is detected
by the monoclonal antibody 8H9. This antigen is expressed on a
broad spectrum of human neuroectodermal, mesenchymal and epithelial
tumors and appears to be immunohistochemically tumor specific,
namely, it is expressed on cell membranes of tumor cells with
no/low membrane reactivity noted on normal human tissues. The
antigen was present on 88% of neuroectodermal tumors, 96% of
mesenchymal tumors and 44% of epithelial cancers tested. The
specific tissue distribution suggests a unique tumor antigen not
previously reported.
The expression of the 8H9 antigen on several glial and nonglial
brain tumors and the complete absence on normal brain tissue is
unusual. This property contrasts with most of the previously
described glial tumor antigens with a cell membrane distribution
(Table 5). Neuroectodermal-oncofetal antigens e.g. neural cell
adhesion molecules are present to varying degrees on normal adult
and fetal tissues (6). Neurohematopoeitic antigens including Thy-1
determinants (24), CD-44 (8) and its splice variants (25) are
present on normal and neoplastic brain tissue as well as
hematopoeitic tissues, principally of the lymphoid lineage.
Gangliosides, such as G.sub.D2 and G.sub.M2, although expressed on
tumors of neuroectodermal origin, are also present on normal brain
tissue (7). The lactotetraose series ganglioside 3'-6''-iso
L.sub.D1 is widely expressed on brain tumors and on epithelial
cancers and germ cell tumors as well as on normal brain tissue.
(26).
TABLE-US-00005 TABLE 5 Antigens expressed on glial tumors Antigen
Antibody Crossreactivity with normal tissues Cell Membrane antigens
Neurohematopoeitic antigens Thy-1 Ab 390 (24) Normal neuronal cells
CD44 Multiple Normal endothelium CD44 splice variants Multiple (25)
Normal neuronal cells Cell Adhesion molecules NCAM ERIC-1 (11),
UJ13-A (10) Normal neuronal cells Normal neuronal cells Integrin 3
ONS-M21 (30) Not reactive with normal brain Gangliosides G.sub.D2
3F8 (35) Normal neuronal cells 3'-6' iso-LD1 DMAb-22 (29) Fetal
brain, reactive astrocytes Growth Factor Receptors EGFRvIII MR1 (9)
No normal tissues; breast and lung carcinoma PDGFR- Anti-PDGFR-7
(36) Normal neuronal cells Uncharacterized Ependymoma-associated
MabEp-C4 (34) Not reactive with PBL, normal brain Glioma-associated
GA-17, GB-4, GC-3 (32) Not reactive with normal adult or fetal
brain Glioma-associated 6DS1 (33) Not reactive with normal adult or
fetal brain Intracellular IFAP-300 Anti-IFAP-300 kDa (37) Not
reactive with normal brain GFAP Multiple Normal neuronal cells
Interstitial matrix Tenascin 81C6 (13), Normal liver, kidney; not
reactive with adult brain BC-2 (14) Not reactive with normal brain
GP-240 Mel-14 (38) Melanoma; not reactive with normal brain
Oncofetal fibronectin BC-1 (39) Adult endometrium; not reactive
with normal brain
Another remarkable property of the 8H9 antigen is its expression on
tumors of diverse lineage: neuroectodermal, mesenchymal and to a
lesser degree epithelial tumors. No monoclonal antibody to date has
the binding spectrum described with 8H9. This broad distribution
provides MoAb 8H9 the potential of being a "generic" tumor antigen
for targeted therapy. Of particular interest is its expression on
28/29 rhabdomyosarcoma tumors and the rhabdomyosarcoma cell lines
tested by indirect immunofluorescence. Disseminated and high risk
rhabdomyosarcomas have a very poor prognosis with <40% long term
survival rate (27). Although the MYOD family of oncofetal proteins
are specific to rhabdomyosarcoma, they are nuclear antigens and
therefore unlikely candidates for antibody-based therapy (19). In a
preliminary report, cross reactivity of the monoclonal antibody
BW575 raised against small cell lung carcinoma with
rhabdomyosarcoma cell lines and 2/2 rhabdomyosarcoma sections was
described. However, this antibody showed cross-reactivity with
normal tissues (28).
Two further groups of tumors studied were the Ewing's family of
tumors and osteosarcoma. The Ewing's family of tumors can be
differentiated from other small blue round cell tumors of childhood
by monoclonal antibodies recognizing glycoprotein p30/32 coded by
MIC2 oncogene. However, this protein is also expressed on normal
tissues and on other tumors, severely limiting its utility in
radioimaging and therapy (18). 100% (21/21) of Ewing's family
tumors tested showed immunoreactivity with MoAb 8H9. Apart from
G.sub.D2 (15), the osteosarcoma-associated antigen recognized by
the MoAbs TP-1 and TP-3 (17), and the decay accelerating factor
CD55 (16), few tumor-associated antigens have been defined for
osteosarcoma. In our study 28/29 (95%) osteosarcomas tested
immunohistochemically positive with MoAb 8H9. The latter may
therefore have clinical utility in the Ewing's family of tumors and
osteosarcomas.
The 8H9 antigen appears to be a novel, previously undescribed
antigen. Sensitivity to proteinase suggests that it has a protein
component. Conversely, the lack of sensitivity to neuraminidase
implies absence of sialic acid residues, and the lack of
sensitivity to phosphatidyl-inositol specific phospholipase C
implies that the 8H9 antigen is not GPI anchored. It is unlikely to
be related to the neural cell adhesion molecule family due to its
unique distribution and restriction of expression among normal
tissues (6). Of the currently described antibodies, which bind to
glial tumors, four have been reported to be restricted to tumor
tissues. The mutated EGFRvIII was found to be expressed on 52% of
gliomas tested and crossreacts with breast and lung carcinomas
(29). However, the broad distribution of the 8H9 antigen is
different from EGFRvIII. Integrin 3, a 140 kDa protein expressed on
gliomas and medulloblastomas is targeted by the monoclonal antibody
ONS-M21 which does not cross react with normal brain (30). However,
negative immunoreactivity with neuroblastoma, melanoma and
meningioma has been reported. (31). Similar data on glioma-specific
antibodies with no cross reactivity with normal brain has been
published. However, they do not react with other neuroectodermal or
mesenchymal tumors and data regarding reactivity with other tissues
is unavailable (32). A 38 kDa antigen has been targeted on
glioblastoma cells by the antibody 6DS1. No crossreactivity with
human brain has been reported. Data regarding reactivity with other
human tissues is unknown, although a high accumulation of the
radiolabeled antibody in mouse kidney has been reported. (33). An
ependymoma-specific protein antigen of 81 kDa, recognized by
monoclonal antibodies which do not crossreact with normal glial
cells, has also been described. These antibodies do not react with
other glial tumors such as glioblastoma and crossreactivity with
other tumor tissue is not known (34).
The homogeneous expression of the 8H9 antigen on cell membrane
makes it an attractive candidate for targeted immunotherapy.
Furthermore, the persistence of the 8H9 antigen on NMB7 cells after
binding to the MoAb suggests that the antigen is not easily
immunomodulated. In order to explore its potential for radioimaging
we used .sup.99mTc conjugated 8H9 to image neuroblastoma xenografts
in athymic nude mice. This revealed selective uptake in the
xenografts apart from moderate uptake in the liver, % ID/gm being
50% of that achieved with the anti-G.sub.D2 monoclonal antibody 3F8
(data not shown). The hydrazino-derivative of 8H9, therefore,
retains the immunoreactive properties of the unmodified antibody,
and may be useful for radioimaging of tumors. We have also
demonstrated selective radioimmunolocalization of rhabdomyosarcoma
xenografts in athymic mice with no significant uptake in normal
tissues using .sup.125I-labeled 8H9 (data not shown).
In summary, the monoclonal antibody 8H9 recognizes a unique 58 kD
tumor-specific antigen with broad distribution across a spectrum of
tumors of varying lineage: neuroectodermal, mesenchymal and
epithelial, with restricted expression in normal tissues. 8H9 may
have clinical utility in the targeted therapy of these human solid
tumors in vitro or in vivo. Further biochemical characterization of
the 8H9 antigen is warranted and may be of interest in delineating
a possible role in the oncogenic process.
REFERENCES
1. Cheung, N. K. V., Kushner, B. H., Cheung, I. Y., Canete, A.,
Gerald, W., Liu, C., Finn, R., Yeh, S. J., Larson, S. M. Anti-GD2
antibody treatment of minimal residual stage 4 neuroblastoma
diagnosed at more than 1 year of age. J. Clin. Oncol.,
16:3053-3060, 1998. 2. Yu, A., Uttenreuther-Fischer, M., Huang,
C.-S., Tsui, C., Gillies, S., Reisfeld, R., Kung, F. Phase I trial
of a human-mouse chimeric anti-disialoganglioside monoclonal
antibody ch14.18 in patients with refractory neuroblastoma and
osteosarcoma. J. Clin. Oncol., 16:2169-2180, 1998. 3. Jurcic, J.
G., Caron, P. C., Miller, W. H., Yao, T. J., Maslak, P., Finn, R.
D., Larson, S. M., Warrell, R. P. J., Scheinberg, D. A. Sequential
targeted therapy for acute promyelocytic leukemia with all-trans
retinoic acid and anti-CD33 monoclonal antibody M195. Leuk.,
9:244-248, 1995. 4. Pegram, M. D., Slamon, D. J. Combination
chemotherapy with trastuzumab (Herceptin) and cisplatin for
chemoresistant metastatic breast cancer: evidence for
receptor-enhanced chemosensitivity. Sem. Oncol., 26:89-95, 1999. 5.
Czuczman, M. S., Grilo-Lopez, A. J., White, C. A., Saleh, M.,
Gordon, L., LoBuglio, A. F., Jonas, C., Klippenstein, D., Dallaire,
B., Yarns, C. Treatment of patients with low-grade B-cell lymphoma
with the combination of chimeric anti-CD20 monoclonal antibody and
CHOP chemotherapy. J. Clin. Oncol., 17:268-276, 1999. 6.
Garin-Chesa, P., Fellinger, E. J., Huvos, A. G., Beresford, H. R.,
Melamed, M. R., Triche, T. J., Rettig, W. J. Immunohistochemical
analysis of neural cell adhesion molecules. Differential expression
in small round cell tumors of childhood and adolescence. Am. J.
Pathol., 139:275-286, 1991. 7. Ritter, G., Livingston, P. O.
Ganglioside antigens expressed by human cancer cells. Semin.
Cancer. Biol., 2:401-409, 1991. 8. Ylagan, Quinn, L. R. B: CD44
expression in astrocytic tumors. Modern Pathology, 10:1239-1246,
1997. 9. Kuan, C. T., Reist, C. J., Foulon, C. F., Lorimer, I. A.,
Archer, G., Pegram, C. N., Pastan, I., Zalutsky, M. R., Bigner, D.
D. 125I-labeled anti-epidermal growth factor receptor vIII
single-chain Fv exhibits specific and high-level targeting of
glioma xenografts. Clin. Can. Res., 5:1539-1549, 1999. 10.
Richardson, R. B., Davies, A. G., Bourne, S. P., Staddon, G. E.,
Jones, D. H., Kemshead, J. T., Coakham, H. B.
Radioimmunolocalization of human brain tumors. Biodistribution of
radiolabelled monoclonal antibody UJ13A. Eur J Nucl Med,
12:313-320, 1986. 11. Papanastassiou, V., Pizer, B. L., Coakham, H.
B., Bullimore, J., Zananiri, A., Kemshead, J. T. Treatment of
recurrent and cystic malignant gliomas by a single intracavitary
injection of 131I-monoclonal antibody: Feasibility,
pharmacokinetics and dosimetry. Br. J. Cancer, 67:144-151, 1993.
12. Celis, E., Tsai, V., Crimi, C., Demars, R., Wentworth, P. A.,
Chesnut, R. W., Grey, H. M., Sette, A., Serra, H. M. Induction of
anti-tumor cytotoxic T lymphocytes in normal humans using primary
cultures and synthetic peptide epitopes. Proc. Natl. Acad. Sci.
USA, 91:2105-2109, 1994. 13. Bigner, D. D., Brown, M. T., Friedman,
A. H., Coleman, R. E., Akabani, G., Friedman, H. S., Thorstad, W.
L., Mclendon, R. E., Bigner, S. H., Zhao, X. G. Iodine-131-labeled
antitenascin monoclonal antibody 8106 treatment of patients with
recurrent malignant gliomas: phase I trial results. Journal
Clinical Oncology, 16:2202-2212, 1998. 14. Riva, P., Frnceschi, G.,
Frattarelli, M., Riva, N., Guiduci, G., Cremonini, A. M., Giuliani,
G., Casi, M., Gentile, R., Jekunen, A., Kairemo, K. J. 1311
radioconjugated antibodies for the locoregional radioimmunotherapy
of high-grade malignant glioma-phase I and II study. Acta Oncol,
38:351-359, 1999. 15. Heiner, J., Miraldi, F. D., Kallick, S.,
Makley, J., Smith-Mensah, W. H., Neely, J., Cheung, N. K. V. In
vivo targeting of GD2 specific monoclonal antibody in human
osteogenic sarcoma xenografts. Cancer Res., 47:5377-5381, 1987. 16.
Spendlove, I., James, L. L., Carmichael, J., Durrant, L. G. Decay
accelerating factor (CD55): a target for cancer vaccines? Cancer
Res., 59:2282-2286, 1999. 17. Bruland, O., Fodstad, O., Funderud,
S., Pihl, A. New monoclonal antibodies specific for human sarcomas.
Int J Cancer, 15:27-31, 1986. 18. Weidner, N., Tjoe, J
Immunohistochemical profile of monoclonal antibody O13 that
recognizes glycoprotein 930/32MIC2 and is useful in diagnosing
ewing's sarcoma and peripheral neuroepithelioma. American Journal
of Surgical Pathology, 18:486-494, 1994. 19. Wang, N. P., Marx, J.,
McNutt, M. A., Rutledge, J. C., Gown, A. M. Expression of myogenic
regulatory proteins (myogenin and MyoD1) in small blue round cell
tumors of childhood. Am. J. Pathol., 147:1799-1810, 1995. 20.
Hatzubai, A., Maloney, D. G., Levy, R. The use of a monoclonal
anti-idiotype antibody to study the biology of human B-cell
lymphoma. J. Immunol., 126:2397-2402, 1981. 21. Cheung, N. K.,
Saarinen, U., Neely, J., Landmeier, B., Donovan, D., Coccia, P.
Monoclonal antibodies to a glycolipid antigen on human
neuroblastoma cells. Cancer Res., 45:2642-2649, 1985. 22. Kramer,
K., Gerald, W., LeSauteur, L., Saragovi, H. U., Cheung, N. K. V.
Prognostic value of TrkA protein detection my monoclonal antibody
5C3 in Neuroblastoma. Clin. Can. Res., 2:1361-1367, 1996. 23.
Hecht, T. T., Longo, D. L., Cossman, J., Bolen, J. B., Hsu, S.-M.,
Israel, M., Fisher, R. I. Production and characterization of a
monoclonal antibody that binds reed-sternberg cells. J. Immunol.,
134:4231-4236, 1985. 24. Seeger, R. C., Danon, Y. L., Rayner, S.
A., Hoover, F. Definition of a Thy-1 determinant on human
neuroblastoma, glioma, sarcoma, and teratoma cells with a
monoclonal antibody. J. Immunol., 128:983-989, 1982. 25. Kaaijk,
P., Troost, D., Morsink, F., Keehnen, R. M., Leenstra, S., Bosch,
D. A., Pals, S. T. Expression of CD44 splice variants in human
primary brain tumors. Journal of Neuro-Oncology, 26:185-190, 1995.
26. Wikstrand, C. J., Longee, D. C., McLendon, R. E., Fuller, G.
N., Friedman, H. S., Fredman, P., Svennerholm, L., Bigner, D. D.
Lactotetraose series ganglioside 3',6'-isoLD1 in tumors of central
nervous and other systems in vitro and in vivo. Cancer Res.,
53:120-126, 1993. 27. Pappo, A., Shapiro, D. N., Crist, W. M.
Rhabdomyosarcoma: biology and treatment. Pediatr. Clin. North Am.,
44:953-972, 1997. 28. Fujisawa, T., Xu, Z. J., Reynolds, C. P.,
Schultz, G., Bosslet, I. V., Seeger, R. C. A monoclonal antibody
with selective immunoreactivity for neuroblastoma and
rhabdomyosarcoma. Proc. Am. Assoc. Cancer Res., 30:345, 1989. 29.
Wikstrand, C. J., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey,
P. A., Kurpad, S. N., McLendon, R. E., Moscatello, D., Pegram, C.
N., Reist, C. J., et al. Monoclonal Antibodies against EGFRvIII are
Tumor Specific and React with Breast and Lung Carcinomas and
Malignant Gliomas. Cancer Res., 55:3140-48, 1995. 30. Kishima, H.,
Shimizu, K., Tamura, K., Miyao, Y., Mabuchi, E., Tominage, E.,
Matsuzaki, J., Hayakawa, T. Monoclonal antibody ONS-21 recognizes
integrin a3 in gliomas and gliomas and medulloblastomas. Br. J.
Cancer, 79:333-339, 1998. 31. Moriuchi, S., Shimuzu, K., Miyao, Y.,
Hayakawa, T. Characterization of a new mouse monoclonal antibody
(ONS-M21) reactive with both medulloblastomas and gliomas. Br. J.
Cancer, 68:831-837, 1993. 32. Kondo, S., Miyatake, S., Iwasaki, K.,
Oda, Y., Kikuchi, H., Zu, Y., Shomoto, M., Namba, Y. Human
glioma-specific antigens detected by monoclonal antibodies.
Neurosurgery, 30:506-511, 1992. 33. Dastidar, S. G., Sharma, S. K.
Monoclonal antibody against human glioblastoma multiforme (U-87Mg)
immunoprecipitates a protein of monoclonal mass 38 KDa and inhibits
tumor growth in nude mice. J Neuroimmuno, 56:91-98, 1995. 34.
Mihara, Y., Matsukado, Y., Goto, S., Ushio, Y., Tokumitsu, S.,
Takahashi, K. Monoclonal antibody against ependymoma-derived cell
line. Journal of Neuro-Oncology, 12:1-11, 1992. 35. Daghighian, F.,
Pentlow, K. S., Larson, S. M., Graham, M. C., DiResta, G. R., Yeh,
S. D., Macapinlac, H., Finn, R. D., Arbit, E., Cheung, N. K.
Development of a method to measure kinetics of radiolabeled
monoclonal antibody in human tumour with applications to
microdosimetry: positron emission tomography studies of iodine-124
labeled 3F8 monoclonal antibody in glioma. Eur J Nucl Med,
20:402-409, 1993. 36. Plate, K. H., Breier, G., Farell, C. L.,
Risau, W. Platelet derived growth factor b is induced during tumor
development and upregulated during tumor progression in endothelial
cells in human gliomas. Lab. Invest., 67:529-534, 1992. 37. Yang,
H. S., Lieska, N., Glick, R., Shao, D., Pappas, G. D. Expression of
300-kilodalton intermediate filament-associated protein
distinguishes human glioma cells from normal astrocytes.
Proceedings of the National Academy of Sciences of the United
States of America, 90:8534-8537, 1993. 38. Bigner, D. D., Brown,
M., Coleman, E., Friedman, H. A., McClendon, R. E., Bigner, S. H.,
Zhao, X. G., Wikstrand, C. J., Pegram, C. N. Phase I studies of
treatment of malignant gliomas and neoplastic meningitis with 131 I
radiolabeled monoclonal antibodies anti-tenascin 8106 and
anti-chondroitin proteoglycan sulfate MeI-14 (ab').sub.2-- a
preliminary report. J Neuro Oncol, 24:109-122, 1995. 39. Mariani,
G., Lasku, A., Pau, A., Villa, G., Motta, C., Calcagno, G., Taddei,
G. Z., Castellani, P., Syrigos, K., Dorcaratto, A., et al. A pilot
pharmacokinetic and immunoscintigraphic study with the
technetium-99m-labeled monoclonal antibody BC-1 directed against
oncofetal fibronectin in patients with brain tumors. Cancer
Supplement, 80:2484-2489, 1997.
Second Series of Experiments
Recent clinical trials have shown promising potentials of
monoclonal antibodies (MoAbs) in the treatment of cancer: anti-CD20
(lymphoma), anti-HER2 (breast cancer), anti-tenascin (brain
tumors), anti-CD33 (leukemia), and anti-TAG-72 (colon cancer). In
pediatric oncology, tumor-targeting agents are even more relevant
since minimal residual disease (MRD) is often the obstacle to cure,
and late effects of non-specific therapy are significant. Despite
high-intensity combination therapy, most metastatic solid tumors
(Ewing's sarcoma [ES], primitive neuroectodermal tumor [PNET],
osteosarcoma [OS], desmoplastic small round cell tumor [DSRT],
rhabdomyosarcoma [RMS], and brain tumors) remain incurable. Using
metastatic neuroblastoma (NB) for proof of principle, our
laboratory integrated the murine IgG3 anti-ganglioside GD.sub.2
MoAb 3F8 into multi-modality therapy. 3F8 has demonstrated high
selectivity and sensitivity in radioimmunodetection of metastatic
tumors, and appears to be a safe and effective method of
eliminating MRD, achieving a >50% progression-free survival
(PFS). For most pediatric solid tumor therapeutic MoAbs do not
exist. Known tumor surface antigens are often restricted to a
specific tumor type, heterogeneous in its expression, or found in
normal blood cells or organs. We recently described a MoAb 8H9
which recognizes a novel cell surface antigen in a wide spectrum of
pediatric tumors, with no crossreactivity with blood, marrow, brain
and normal organs, and minimal reactivity with hepatocyte
cytoplasm. .sup.131I or .sup.99mTc-labeled 8H9 can effectively
image NB and RMS xenografts in SCID mice. Antigen expression was
generally homogeneous within tumors, and did not modulate on MoAb
binding. We propose to test the targeting potential of
.sup.131I-8H9 in a pilot imaging study. Pediatric/adolescent
patients with NB, RMS, ES, PNET, OS, DSRT and brain tumors are
subjects of our investigation. We have two specific aims:
#1: To measure the level of agreement between conventional imaging
modality (CT, MRI, and nuclear scans) and antibody 8H9 imaging in
known and occult sites of disease. Sensitivity analysis of 8H9 for
each disease type will be conducted.
#2: To calculate the absorbed dose delivered by .sup.131I-8H9 to
tumor relative to normal organs.
Background and Significance
MoAb selective for tumors have therapeutic potential .sup.1,2 The
introduction of hybridoma technology by Kohler and Milstein in
1975.sup.3 and the advances in molecular biologic techniques have
greatly expanded the potential of MoAb in human cancers. Optimal
targeting of MoAb demands high tumor antigen density with
homogeneous expression, lack of antigen modulation on tumor cell
surface, adequate vascularity of tumor to allow deep penetration,
minimal toxicity on normal tissues, low reticulo-endothelial system
(RES) uptake, noninterference by circulating free antigens, and low
immunogenicity. In practice, very few MoAb-antigen-tumor model
systems have fulfilled these stringent criteria. Recent clinical
trials have shown promising potentials of MoAbs. Anti-CEA antibody
in colorectal cancer,.sup.4, anti-CD20 antibodies in
lymphoma,.sup.5 anti-HER2 antibodies in breast cancer,.sup.6
anti-tenascin antibodies in glial brain tumors,.sup.7 MoAb M195
against CD33 in acute leukemia.sup.8 and anti-TAG-72 antibodies in
colon cancer.sup.9 have demonstrated efficacy in clinical trials.
Our laboratory has developed the MoAb 3F8 which targets the
ganglioside G.sub.D2 overexpressed on NB. 3F8 has been shown to
have a high specificity and sensitivity in the radioimmunodetection
of minimal residual disease (MRD) in patients with NB,.sup.10 and a
significant impact when used as adjuvant therapy..sup.11 131I has
been a common isotope used both for imaging and therapy purposes.
Although not widely available, pure-emitters such as
.sup.90Y,.sup.12,13 alpha-emitting particles,.sup.14,15 such as
.sup.211At, .sup.212Bi and .sup.213Ac have attractive properties
with promising biological effectiveness. Multiple radioisotopes of
varying path lengths and half-lives may be needed to enhance
radiocurability of both bulk and microscopic diseases. More recent
developments in immunocytokines (e.g. IL-2, IL-12),.sup.16
bispecific antibodies for pretargeting strategies (e.g.
radioisotopes or drugs),.sup.17,18 or T-bodies for retargeting
immune cells.sup.19-21 have further expanded the potentials of
antibody-based immunotherapies.
Brain tumor antigens Examples of tumor antigens expressed on glial
tumors include neuroectodermal-oncofetal antigens eg. neural cell
adhesion molecules (NCAM),.sup.22 gangliosides (GD2, GM2,
3'-6''-iso LD1).sup.23,24 and neurohematopoeitic antigens (Thy-1,
CD44 and splice variants)..sup.25-27 All of these antigens are
present to varying degrees on normal adult and fetal tissues, and
for some hematopoeitic tissues as well. Notwithstanding the
universal expression of NCAM by neuronal cells, anti-NCAM MoAb
UJ13A was shown to accumulate in gliomas by virtue of disruption of
blood brain barrier locally.sup.28 and another MoAb ERIC-1 showed
clinical benefit in resected glioma cavities..sup.29 Integrin-3, a
140 kDa protein expressed on gliomas and medulloblastomas and not
in normal brain, is a potential target (MoAb ONS-M21).sup.30, but
it is poorly expressed among other tumor types..sup.31 The
extracellular matrix protein tenascin is expressed in 50-95% of
gliomas as well as on mesenchymal tumors, carcinomas, normal human
glial, liver and kidney cells..sup.32 Anti-tenascin monoclonal
antibodies 81C6,.sup.7 BC-2 and BC-4.sup.33 administered directly
into tumor-cavities have shown efficacy in patients with malignant
gliomas. More recent investigations have focused on growth factor
receptors. in particular type III mutant epidermal growth factor
receptor (EGFRvIII) expressed on 52% of gliomas.sup.34 as well as
breast and lung carcinomas..sup.35 Given the relationship of these
mutated receptors to their malignant potential, they may be ideal
targets for MoAb. Although other glioma-specific antibodies with no
cross reactivity with normal brain have been described (e.g. 6DS1,
MabEp-C4),.sup.36-38 they have limited reactivity with other
neuroectodermal or mesenchymal tumors, and data regarding
cross-reactivity with normal tissues are not available. To date,
with the exception of EGFRvIII, the glial tumor antigens described
are either found on normal brain and/or normal tissues, restricted
to specific tumor types, or found in intracellular
compartments/extracellular matrix which can limit their clinical
utility for targeting to single cells or spheroids.
Sarcoma antigens Optimal tumor antigens, similarly, have not been
defined for the large family of sarcomas. Although the MyoD family
of oncofetal proteins are specific to rhabdomyosarcoma, they are
localized to the nucleus and therefore do not offer targets for
antibody-based therapy..sup.39 The ES family of tumors can be
differentiated from other small blue round cell tumors of childhood
by MoAbs recognizing glycoprotein p30/32 coded by the MIC2
oncogene. However, this protein is expressed on normal tissues
(e.g. T-cells).sup.40 greatly limiting the utility of MoAb in
marrow purging, radioimaging or radiotherapy..sup.41 glycoprotein
p72,.sup.43 CD55.sup.44 erB2/neu.sup.45 and the antigen recognized
by the MoAb TP-3..sup.46 CD55 is decay-accelerating factor, a
ubiquitous protein on blood cells and most tissues to prevent
complement activation. Clearly MoAb directed at CD55 would have
significant limitations for in vivo targeting. The degree of tumor
heterogeneity (e.g. erbB2 in OS) may also limit the efficacy of
MoAb-targeted approach. The presence of GD2 on pain fibers causes
significant pain side effects in clinical trials. Nevertheless,
this side effect is self-limited and this cross-reactivity did not
interfere with the biodistribution and clinical efficacy of
specific MoAb (see preliminary results). Nevertheless, GD2 is
generally low or absent in RMS, ES, PNET, and many soft-tissue
sarcomas. In addition, the presence of GD2 in central neurons can
limit its application in tumors arising or metastatic to the brain.
Our laboratory has generated a novel MoAb 8H9 by hyperimmunizing
female BALB/c mice with human NB..sup.47 8H9 recognizes a unique
surface antigen homogeneously expressed on cell membranes of a
broad spectrum of tumors of neuroectodermal, mesenchymal and
epithelial origin, with restricted distribution on normal tissues
(see preliminary results)..sup.48
The availability of an antibody with broad specificity for
pediatric tumors will facilitate several lines of clinical
investigations. In vitro, such antibodies will be extremely useful
for (1) detecting lymph node or marrow metastasis,.sup.49 (2)
enrichment/isolation of circulating tumor cells for RT-PCR
detection strategies,.sup.50 (3) purging of bone marrow before
autologous bone marrow transplantation,.sup.51 (4) purging of ex
vivo activated T-cells prior to adoptive cell therapy. In vivo its
utility can go beyond its diagnostic capability. When chimerized
with a human-1Fc tail, it becomes tumoricidal through
complement-mediated, and antibody-dependent cell-mediated
cytotoxicities..sup.52 Through single-chain Fv constructs, new
fusion proteins can now be delivered to tumor sites (e.g. IL-2,
IL-12, toxins, or enzymes). Bivalent scFv and tetravalent scFv can
be engineered to improve avidity..sup.53 Bispecific scFv can be
constructed to engage cells and proteins in various targeting
strategies (e.g. pretargeting)..sup.17,18 ScFv can also be used in
T-bodies to retarget T-cells, a powerful technique to increase
clonal frequency and bypassing the HLA requirement of TCR
functions..sup.19-21 Furthermore scFv-fusion proteins (e.g. CD28,
zeta chain) transduced into T-cells can greatly enhance their
survival following activation..sup.21 Even more importantly, the
ability of such cells to proliferate in contact with tumor cells
can further amplify the efficiency of T-cell cytotherapy.
Radioimmunoscintigraphy can test if an antibody-antigen system has
targeting potential. Using radioiodines and technetium we have
demonstrated the utility of the GD2 system for targeting in the
last decade. This information has been translated into treatment
strategies using both unlabeled and .sup.131I labeled antibody 3F8.
Dosimetry calculations have allowed quantitative estimates of
therapeutic index when cytotoxic agents are delivered through
antibody-based methods. Uptake (peak dose and area under the curve
AUC) in specific organs relative to tumor can be measured. These
studies are resource intensive and to be done well, require
laboratory, radiochemistry, nuclear medicine, medical physics and
clinical resource support, as well as substantial personnel effort.
In pediatric patients, issues of therapeutic index may be even more
pressing given the potential of late effects of treatment. In
addition, despite the potential life-years saved for pediatric
cancer, orphan drugs are not economically attractive for most
industrial sponsors. These circumstances have made the initial
stages of clinical development even more stringent and relatively
more difficult to accomplish.
Patient monitoring and correlative laboratory studies
Pharmacokinetic studies are crucial in our understanding of
antibody targeting, its toxicity and its efficacy.
Radioimmunoscintigraphy uses the trace label principle and gamma
imaging to define the distribution of a specific antibody in
various human organs. It provides estimates of antibody (and
radiation) dose delivered to blood, marrow and major organs. The
continual development of improved software and hardware for
calculating antibody deposits in tissues is critical in
implementing these studies (see preliminary results). The
quantitative relationship of free circulating antigens (if present)
and biodistribution of MoAb needs to be defined. The formation of
human-anti-mouse antibody (HAMA) response will clearly affect the
in vivo properties of these antibodies. However, the induction of
the idiotype network (see preliminary results) may have potential
benefit in the long run. These parameters need to be monitored.
These in vitro assays will provide important information in
understanding and optimization of future use of 8H9 and other MoAb
in the context of chemo-radiotherapy for a broad category of
recalcitrant tumors in children, adolescents and young adults.
Memorial Sloan-Kettering Cancer Center (MSKCC) is devoted to the
research and clinical care of cancer patients. The Center has an
extensive patient referral base, particularly within the tri-state
area. The center has an established commitment and past record in
the use of monoclonal antibodies in the diagnosis and therapy of
human cancers, including melanoma, colon cancer, and leukemias.
Over the past 4 years we have an annual accrual of around 45 new
NB, 27 OSs, 58 brain tumors, 23 Ewing's/PNET, 18 retinoblastoma, 12
rhabdomyosarcomas, 16 sarcomas and 7 DSRT at MSKCC. We are
confident that we can accrue 60 patients within the next 2 years.
In this past decade, we tested the utility of MoAb in the curative
treatment of a lethal tumor (metastatic stage 4 NB in children).
For this orphan disease, the lack of corporate/pharmaceutical
sponsor has made our progress slow and difficult. Nevertheless, we
made the following observations. (1) MoAb can extend the
progression-free period in a cancer that was uniformly lethal two
decades ago. (2) It is feasible to integrate MoAb into standard
chemo-radiotherapy strategies, in order to derive maximal benefit
from all available modalities. (3) Immune based therapies can be
administered safely in the outpatient setting, thus reducing
expensive in-patient costs and maximizing time in the home
environment. (4) MoAb can induce idiotype network, a potential
endpoint that underlies the biology in maintaining continual
clinical remission. (5) GD2 is a useful marker of MRD, and specific
MoAbs are highly efficacious in monitoring and purging of tumor
cells. (6) Novel bioengineering strategies have been developed for
the GD2-3F8 antigen-antibody system which are directly applicable
to other MoAbs (single chain Fv,.sup.54 and T-bodies.sup.55).
During this period, >240 patients have been treated at Memorial
Hospital with the antibody 3F8. A total of >3500 doses of
unlabeled 3F8 have been given, 250 injections of .sup.131I-3F8 for
imaging, and 372 injections of .sup.131I-3F8 for therapy. Although
there were side effects, there were no lethal sequella during or
immediately after antibody administration. 3F8 treatment is now
routinely done in the outpatient clinic. Extending these findings
to a second antigen-antibody system, especially one that will
target to a broader spectrum of pediatric solid tumors is a
priority. The murine IgG1 antibody 8H9 has obvious potential in
monitoring and purging of MRD, radioimmunoscintigraphy, and
radioimmunotherapy (both intravenous or compartmental). If our
proposed study produces favorable results, i.e. selective tumor
uptake at optimal AUC ratios (Tumor: tissues/organs),
radioimmunotherapy can be explored for some of these solid tumors.
More importantly, further development of the antibody would involve
a major effort in humanizing and further genetic engineering to
improve effector functions.
Preliminary Results:
G.sub.D2-specific MoAb-based targeted therapy: a curative approach
to a pediatric solid tumor: metastatic NB Improved understanding of
the biology of NB has reshaped our clinical approach to this
cancer. Non-infant stage 4 NB remains a therapeutic challenge
despite four decades of combination chemotherapy. Similar to many
cancers, MRD state can be achieved in patients with NB after
intensive induction therapy..sup.56,57 Unfortunately, the
transition from MRD to cure was a formidable hurdle..sup.56
Targeted immunotherapy besides being more specific and less toxic,
may supplement what chemoradiotherapy has not
accomplished..sup.58,59 Disialoganglioside G.sub.D2 is a tumor
antigen well suited for targeting therapy because (1) it is
expressed at a high density in human NB, is restricted to
neuroectodermal tissues and is genetically stable, unlike other
tumor antigens such as immunoglobulin idiotypes;.sup.60 (2)
although it circulates in patients' serum, it does not interfere
with the biodistribution of specific antibody (e.g. 3F8), allowing
excellent tumor localization of NBs in patients;.sup.10 (3) it is
not modulated from cell surface upon binding to antibodies; (4) it
is expressed homogeneously in human NB, with little heterogeneity
within tumors and among patients. Several antibodies against
G.sub.D2 antigen has been described (3F8, 14.G2a, 14.18)..sup.47,61
In vitro they can target lymphocytes,.sup.62,63
granulocytes,.sup.52,64,65 complement,.sup.66,67 activated
monocytes/macrophages,.sup.68,69, IL2,.sup.70,
isotopes,.sup.10,59,71,72 toxins,.sup.73,74 and
superantigen..sup.75 Phase I and phase II studies have shown only
modest efficacy,.sup.76-84 marrow disease more likely to respond
than bulky tumors..sup.85 The major side effects included pain,
allergic reactions and neuropathy..sup.78,85 With long followup,
the role of these anti-G.sub.D2 antibodies at the time of MRD
appears promising.
Radiolabeled anti-G.sub.D2 antibody 3F8 3F8 is a murine IgG.sub.3
MoAb directed at the ganglioside G.sub.D2 expressed on human NB
cells. In preclinical studies .sup.131I-3F8 targeted to human NB
xenografts with exceptionally high % ID/gm. Intravenous
.sup.131I-labeled IgG.sub.3 MoAb 3F8 produced a substantial dose
dependent shrinkage of established NB in preclinical studies. Dose
calculations suggested that tumors that received more than 4,200
rads were completely ablated. Marrow suppression was the dose
limiting toxicity. In patient studies, it is not trapped
nonspecifically by the reticuloendothelial system and penetrates
NBs well (0.04 to 0.11% injected dose/gm)..sup.10,86 Because of the
intact blood brain barrier, .sup.131I-3F8 does not normally
localize to brain, spinal cord or penetrate the surrounding
CSF..sup.10,59
.sup.131I-3F8 is more sensitive than conventional modalities,
including metaiodobenzylguanidine (MIBG) in detecting NB in
patients. The biodistribution of .sup.131I-3F8 was studied in 42
patients (2 mCi per patient) with NB..sup.10 Comparison was made
with .sup.131I-MIBG, .sup.99mTc-MDP (technetium-labeled methylene
diphosphonate) bone scan, as well as CT or MRI. .sup.131I-3F8
detected more abnormal sites (283) than .sup.131I-MIBG (138) or
.sup.99mTc-MDP (69), especially in patients with extensive disease.
In 20 patients with soft tissue tumors demonstrated by CT/MRI,
.sup.131I-3F8 detected the disease in 18 of them. Upon surgical
resection, the two .sup.131I-3F8-imaging-negative tumors revealed
ganglioneuroma, one showing microscopic foci of NB. In contrast,
.sup.131I-3F8-imaging-positive tumors were all confirmed as NBs. In
26 patients with evidence of marrow disease by antibody scans,
14/26 had confirmation by iliac crest marrow aspirate/biopsy
examinations. Agreement between the measured tissue radioactivity
and the estimates based on planar scintigraphy validated the
initial dosimetry calculations. The tumor uptake in patients with
NB was 0.08%-0.1% ID/gm. The calculated radiation dose was 36
rads/mCi delivered to NB and 3-5 rad/mCi to blood.
.sup.131I-3F8 differentiated Gliomas from normal brain
tissues..sup.87,88 In 12 patients with brain tumors, 3F8
immunoscintigraphy was compared with .sup.99mTc-glucoheptonate/DTPA
planar imaging, Thallium 201 single photon emission tomography
(SPECT), and .sup.18FDG positron emission tomography (PET). 10/11
malignant gliomas and 1/1 metastatic melanoma showed antibody
localization. No nonspecific uptake in normal brain or CSF was
detected. Average plasma and total body clearance were 20 h and 47
h, respectively. Antibody localization was measured on surgical
specimens and time activity curves were calculated based on
modified conjugate views or PET. Radioactivity uptake in high grade
glioma peaked at 39 h, which then decayed with a half-life of 62 h.
Tumor uptake at time of surgery averaged 3.5% ID/kg and highest
activity by conjugate view method averaged 9.2% ID/kg (3.5 to
17.8).
Both primary and metastatic Small Cell Lung Cancer were detected by
.sup.131I-3F8.sup.89 10 Patients with SCLC were imaged with
.sup.131I-3F8. Five patients previously treated with
chemoradiotherapy were imaged with 2 mCi at the time of recurrence,
while 5 patients were studied with 10 mCi/1.73 m.sup.2 at the time
of diagnosis. No significant toxicities were seen. All 10/10 tumors
showed localization. Precision of localization was confirmed by
comparing SPECT and CT in the 5 patients injected with the 10 mCi
dose. Average half-lives for plasma and total body clearance were
15 h and 58 h, respectively. The tumor to non-tumor ratios appeared
favorable based on the % ID/gm (see below).
TABLE-US-00006 TABLE 3 % ID/kg after .sup.131I-3F8 injection: Day
Small Spinal Large Tumor sampled Heart Bowel Spleen Liver Cord
Bowel Blood Muscle NB 4 1.7 1.7 1.7 2 2.2 2.4 3.1 3.1 SCLC 6 0.4
0.4 0.9 0.4 -- -- -- 0.2 liver Tumor Kidney Lung Bone Ovaries
Adrenal Bladder Stomach Tumor mets NB 3.1 3.6 4 4 5.7 6.7 6.7 40 --
SCLC 0.9 0.5 -- -- 1.6 0.5 -- 2 15
Myeloablative doses of .sup.131I-3F8 are effective for NB with
minimal extramedullary toxicities. Based on the tracer dose
dosimetry, the absorbed doses to liver, spleen, red marrow, lung,
total body and tumor were 537, 574, 445, 454, 499 and 4926 rads,
respectively. The average rad/mCi were 2.3, 2.5, 2, 2, 1.9, and
13.7, respectively. The chemical toxicities of the antibody 3F8
have been studied in phase I.sup.76,77 and phase II
studies..sup.11,90 Acute toxicities included pain, urticaria, fever
and hypotension which were self-limited. The radiological
toxicities of .sup.131I-3F8 were recently defined in a phase I dose
escalation study. (6, 8, 12, 16, 20, 24, and 28 mCi/kg)..sup.91
Among 10 patients (pts) with progressive disease evaluable for
response, 2 cleared the marrow and 2 had partial responses of soft
tissue tumors. Average tumor dose was 150 rad/mCi/kg. Acute
toxicities of .sup.131I-3F8 treatment included pain (20/24) during
the infusion, fever (20/24) and mild diarrhea. All pts developed
grade 4 myelosuppression. 22/24 pts were rescued with cryopreserved
autologous bone marrow; one patient received GM-CSF; one died of
progressive disease before marrow reinfusion. Hypothyroidism
developed in despite thyroid blockade with oral SSKI plus synthroid
or cytomel. In the subsequent phase II study (N7, IRB94-11, FIG.
1), .sup.131I-3F8 was used to consolidate >50 patients at the
end of induction chemotherapy for their stage 4 NB diagnosed after
1 year of age. Except for hypothyroidism, there were no late
effects of .sup.131I-3F8 treatment.
.sup.124I-3F8 PET imaging was first successfully applied to
NB.sup.92 Positron Emission Tomography (PET) can offer advantages
over planar or single photon emission computed tomography (SPECT)
imaging in the quantitation of spatial radioactivity distribution
over time. .sup.124I is a positron emitter with a 4-day half-life.
We have studied the quantitative capability of PET imaging with
.sup.124I,.sup.93 and have used it for scanning of
.sup.124I-labeled antibodies in animals and humans..sup.92,94,95
Using a brain PET scanner (PC4600, Cyclotron Corp.), with a
relatively low resolution (FWHM=1.2 cm), we demonstrated that
quantitation of .sup.124I is possible (range examined was 0.4 to 4
uCi/ml). Studies using .sup.124I in a rat tumor (4 gram) measured
with this PET scanner were within 8% of the ex-vivo measurement.
Subsequently, two patients were studied on this scanner using
.sup.124I-labeled 3F8 antibody..sup.88,92 A 3-compartment model was
used to study the kinetics of the antibody to provide an estimate
of the binding potential of 3F8 antibody for glioma. These
quantitative studies have also allowed us to estimate the radiation
dose to the tumor cell nucleus from low energy Auger
electrons..sup.88 More accurate quantitation of .sup.124I is now
possible with the GE body PET scanner with even higher
resolution.
.sup.131I-3F8 therapy of leptomeningeal cancer.sup.96 While overt
meningeal disease is rapidly fatal, microscopic deposits in the
cranio-spinal axis will spread even if the primary tumor is
eradicated. The potentials of antibody-derived ligands for the
diagnosis and therapy of LM cancer have not been fully explored.
G.sub.D2 is present on a broad spectrum of human tumors including
medulloblastomas, high-grade astrocytomas, PNET, central NBs, small
cell lung cancer, melanoma, sarcomas, leukemia/lymphomas and
peripheral NBs, many of which have LM spread. Clinical trials using
intravenous injections of anti-G.sub.D2 MoAb 3F8 have not
encountered long-term neurotoxicity in patients followed for up to
13 years. Pharmacokinetic studies in rats showed that at least 50%
of intraventricular .sup.131I-3F8 was eliminated by bulk flow. When
human melanoma leptomeningeal xenografts were present, CSF
radioactivity was retained and AUC (area under curve) increased by
1.5 fold. AUC ratios of tumor to CSF, tumor to brain and tumor to
blood were 14, 86, and 64, respectively. These ratios improved to
15, 209 and 97, respectively, if the rats were pretreated with
diuretics. Direct intraventricular administration of 30 mCi of
.sup.131I-3F8 in cynomolgus monkeys did not induce clinical or
histological toxicity. Since G.sub.D2 tissue distribution (CNS and
peripheral) in the cynomolgus monkey is identical to that of human,
the high radiation dose of IT .sup.131I-3F8 (up to 82 Gy) to CSF in
contrast to blood (<2 Gy) may translate into a meaningful
treatment approach. Moreover, serum antibody against the MoAb (AMA)
was 14-22 fold higher than in the CSF, thereby accelerating blood
clearance (reducing blood radiation dose) without affecting CSF
pharmacokinetics.
Intra-CSF .sup.131I-3F8 imaged G.sub.D2-positive LM cancers
successfully in patients. The pilot study included 5 patients who
had a histologically confirmed diagnosis of a malignancy expressing
G.sub.D2 with LM disease refractory to conventional therapies or
for which no conventional therapy exists. Ommaya catheter
placement, patency and CSF flow was evaluated by .sup.111In DTPA
studies. Five patients (ages 1-61 years) with leptomeningeal or
intraventricular melanoma, ependymoma, rhabdoid tumor (n=2) and
retinoblastoma were evaluated. Active disease was identified by MR
scans in 4 of 5 pts, and by positive CSF cytology in 2. Doses of
0.7-1.9 mCi of .sup.131I-3F8 were injected by Ommaya catheter.
Acute side effects included fever (n=2), and headache (n=2) both
treated with tylenol, and one episode of vomiting (n=1). One pt had
an elevated opening CSF pressure that remained increased for 36-48
hours post-injection. There was no appreciable change in WBC,
platelet counts, liver or kidney functions tests or CSF cell counts
in all 5 patients.
The CSF radioactivity biological half-life, distribution of
radioactivity in the craniospinal axis, and dosimetry at plaques of
disease and surrounding normal tissues were determined by
.sup.131I-3F8 Single Photon Emission Tomography (SPECT). Peak CSF
values were achieved generally within the first hour of injection.
The CSF biological half-life was 3-12.9 hours, and was in close
agreement with the SPECT (7.2-13.1 hours). Estimated dose to the
CSF was 14.9-56 cGy/mCi by CSF samples and 15-31 cGy/mCi by SPECT
analysis. Focal areas of tumor uptake were 27-123 cGy/mCi by SPECT
estimates. The radiation dose to the blood was 0.9-1.9 cGy/mCi
based on blood radioactivity measurements. Post-injection
.sup.131I-3F8 SPECT scans showed distribution throughout the
subarachnoid space along the spinal cord down to the level of the
cauda equina by 4 hours, and progressively over the convexity by 24
hours in all patients. Focal .sup.131I-3F8 uptake was demonstrated
in the ventricles, spine and midbrain in 4 patients, corresponding
to disease seen on MR. In the one patient who had no MR evidence of
disease, .sup.131I-3F8 clearance was most rapid (3 hours), with no
focal accumulation observed on SPECT. Four patients with focal
.sup.131I-3F8 uptake received 10 mCi of .sup.131I-3F8 through the
Ommaya reservoir as part of a treatment protocol in a phase I
toxicity study. Except for grade 2 toxicities (fever, headache,
nausea and vomiting, increase in intracranial pressure) and a
breakthrough seizure, there were no adverse side effects during
their initial treatment. One patient had a radiographic and
clinical response. On repeat treatment 2 months later, with the
same dose, a rapid rise of intracranial pressure necessitated a
shunt placement. Although all 4 treated patients progressed, 3 are
still alive (2+, 3+ and 9+ months from treatment).
Adjuvant anti-G.sub.D2 antibody 3F8 3F8 (without radioisotope) has
also been tested in phase I and phase II studies..sup.58,76,77
Responses of metastatic NB in the bone marrow were seen. Another
mouse antibody 14.G2a and its chimeric form 14.18 have also induced
marrow remissions in patients with NB..sup.83 Acute self-limited
toxicities of 3F8 treatment were pain, fever, urticaria,
hypertension, anaphylactoid reactions, as well as decreases in
blood counts and serum complement levels, and in rare patients
self-limited neuropathy..sup.71,97-99
Anti-GD2 antibody treatment of MRD in stage 4 NB diagnosed at more
than one year of age..sup.11 Thirty-four patients (pts) were
treated with 3F8 at the end of chemotherapy. Most had either bone
marrow (31 pts) or distant bony metastases (29 pts). Thirteen pts
were treated at second or subsequent remission (group I), and 12
pts in this group had a history of progressive/persistent disease
after ABMT; 21 pts (all on N6 protocol) were treated in first
remission following induction chemotherapy (group II). At the time
of 3F8 treatment, all 34 patients had stable or minimal NB.
Twenty-three patients were in CR, 8 in VGPR, 1 PR and 2 with
histological evidence of marrow disease. Since microscopic occult
NB could escape detection by conventional radiographic studies,
three additional sensitive methods were used to document disease
prior to 3F8 treatment. They were 131I-3F8 immunoscintigraphy,
marrow immunocytology, and molecular detection of marrow GAGE by
RT-PCR. Fourteen of 34 patients were 131I-3F8 scan-positive prior
to 3F8 treatment. Nine had residual disease in their marrow by
immunocytology and 12 had evidence of marrow disease by RT-PCR. A
total of 25/34 patients were positive for disease by at least one
of these three methods. Thirteen patients are progression-free (40
to 148+ months from the initiation of 3F8 treatment); one other
patient is alive with disease 61+ months after 3F8 treatment. Both
group I and group II patients achieved long-term progression-free
probabilities of 38%. Among the 20 patients whose disease
progressed after 3F8, 3 in group II had isolated relapse in the
CNS, a sanctuary site where antibody 3F8 could not penetrate.86
Although the majority of patients were in CR/VGPR by conventional
criteria right before 3F8 treatment, 74% had evidence of disease by
the more sensitive methods (immunoscintigraphy with 131I-3F8, bone
marrow immunocytology and RT-PCR). When these tests were repeated
subsequent to 3F8 treatment, 6/9 patients with positive
immunocytology reverted to undetectable. Among the 12 GAGE-positive
patients, 7 became negative for GAGE expression. Six patients had
post-3F8 treatment 131I-3F8 scans and all 6 showed resolution or
improvement.
Human anti-mouse antibody response (HAMA) and patient outcome:
Three patterns of HAMA response were identified. In pattern I, HAMA
was not detectable during the 4-6 month followup period after first
cycle of 3F8, 42% had no HAMA response even after receiving 2-4
cycles of 3F8 over a 4-25 month period. In pattern II, HAMA was
detected but rapidly became negative during the 4-6 month followup
period. In pattern III, HAMA titer was high (>5000 U/ml) and
persistent during the 4-6 month followup period. When patients
developed HAMA (>1000 U/ml) during a treatment cycle, pain side
effects disappeared. In the absence of HAMA (pattern I) or when
HAMA became negative (pattern II), patients received repeat 3F8
treatments. In the presence of HAMA, subsequent 3F8 treatments had
to be delayed. Thus, patients in group III did not get repeat 3F8
treatment during the first 4-6 months, and had fewer total-cycles
and fewer total-days of 3F8 treatment, while pattern I and II
patients were comparable. Kaplan Meier analysis showed a survival
advantage for those with pattern II HAMA response, i.e. a low
self-limiting HAMA response (73% for pattern II versus 33% for
pattern I, and 18% for pattern III). The probability of survival
among patients with pattern II was significantly better than the
pattern I and III patients combined (p<0.05). For patients
progression-free for at least 12 months after the last cycle of
chemotherapy, those receiving four 3F8 cycles had a PFS probability
double those receiving less than 4 cycles (p=0.08). When patients
with pattern II HAMA response and/or four cycles of 3F8 were
considered as a group (FIG. 4), their survival was significantly
better than the other 20 pts (p<0.001). We interpret these
findings to mean a threshold (four 3F8 cycles, each 10-day cycles)
plus a pattern II HAMA response may be necessary to maintain
permanent tumor control.
Idiotype network is a possible mechanism for long term PFS. Since
the HAMA response was primarily anti-idiotypic (Ab2), we postulate
that the subsequent induction of an idiotype network which included
anti-anti-idiotypic (Ab3) and anti-G.sub.D2 (Ab3') responses may be
responsible for tumor control in patients. Their serum HAMA, Ab3,
and Ab3' titers prior to, at 6, and at 14 months after antibody
treatment were measured by ELISA. Long term PFS and survival
correlated significantly with Ab3' (anti-G.sub.D2) response at 6
months, and with Ab3 response at 6 and 14 months. Non-idiotype
antibody responses (anti-mouse-IgG3 or anti-tumor nuclear HUD
antigen) had no apparent impact on PFS or survival. It appears that
the successful induction of an idiotype network in patients maybe
responsible for long term tumor control and prevention of late
relapse among N6 and N7 patients (FIG. 5). Even among patients
treated on N5 (with ABMT, FIG. 5), all of the survivors of bony and
marrow metastases have had imaging studies with 3F8 and had
detectable idiotype network by ELISA.sup.100; similarly no late
relapses were seen. While N5 and N6 groups had no relapses after
.about.3 years from diagnosis or .about.2 years from 3F8 therapy
(including second remission group), among N7 patients, the relapse
curve has leveled off even earlier, around 2 years from
diagnosis.
Integration of 3F8 treatment into multi-modality therapy: N5, N6
and N7 for stage 4 NB >1 year of age: From 1987 to 1999, N5, N6
and N7 protocols were designed sequentially to test the clinical
importance of dose intensity, 3F8, and .sup.131I-3F8 in consecutive
patients with newly diagnosed stage 4 NB. Most of them had very
high-risk clinical and biologic markers, almost all were
diploid/tetraploid and of unfavorable histopathology. Except for
.sup.131I-3F8 and autologous marrow transplant (ABMT), chemotherapy
and 3F8 are routine outpatient procedures. Evaluations at
sequential endpoints compared favorably with predictions: primary
tumor resectability, overall response, and progression-free
survival (PFS). There were no late relapses after 3.5 years from
diagnosis. For N6 (all survivors past 5 years) 40% are
progression-free; for N7, PFS is projected at 55% (p=0.02 when
compared to N5). Causes of death included disease progression,
secondary leukemia, and isolated CNS relapse. Although toxicities
included hearing loss and hypothyroidism which required correction,
a curative strategy for stage 4 NB appeared to be within reach.
Neuroblastoma, 3F8 and GD2 provided us with the proof of principle
that MoAb may have potential in the permanent eradication of MRD in
the curative treatment of solid tumors in the younger population.
Both RIT and idiotype-network induction are possible with murine
MoAb. We therefore undertook an extensive screening of MoAbs to
identify candidates with a broad reactivity with
pediatric/adolescent solid tumors, that may have similar targeting
potential as the antibody 3F8.
Novel antigen for MoAb targeting to solid tumors in children and
young adults Female BALB/c mice were hyperimmunized with human
neuroblastoma according to previously outlined methods..sup.47
Splenic lymphocytes were fused with SP2/0 mouse myeloma cells line.
Clones were selected for specific binding to neuroblastoma on
ELISA. The 8H9 hybridoma secreting an IgG1 monoclonal antibody was
selected for further characterization after subcloning.
Normal and tumor tissue reactivity of 8H9 antibody Frozen sections
from 315 tumors with histologically confirmed diagnoses of cancer
were analyzed for immunoreactivity with MoAb 8H9. (Tables 5 and 6)
15 histologically normal human tissues and 8 normal monkey tissues
were also analyzed ( ).
TABLE-US-00007 TABLE 5 Neuroectodermal Tumors No. 8H9 positive % NB
87 84 97 Brain Tumors 1. Glial Tumors Glioblastomas multiforme 17
15 88 Mixed Glioma 4 3 -- Oligodendroglioma 11 4 36 Astrocytoma 8 6
75 Ependymoma 3 2 -- 2. Primitive PNET Medulloblastoma 2 2 -- 3.
Mixed Neuronoglial tumor 2 1 -- 4. Other Schwannoma 3 3 --
Meningioma 2 2 -- Neurofibroma 1 1 Melanoma 16 12 75 Ewing's Family
of tumors 21 21 100 TOTAL 177 156 88
TABLE-US-00008 TABLE 6 Mesenchymal Tumors No. 8H9 Reactive %
Rhabdomyosarcoma 26 25 96 Osteosarcoma 26 25 96 Desmoplastic small
round cell tumor 34 32 94 Malignant fibrous histiocytoma 1 1 --
Synovial sarcoma 2 1 -- Leiomyosarcoma 4 4 -- Undifferentiated
sarcoma 2 2 -- TOTAL 95 90 95
TABLE-US-00009 TABLE 7 CARCINOMAS NO. 8H9 Reactive % Breast 12 4 33
Bladder 4 1 -- Adrenal 2 1 -- Stomach 1 1 -- Prostate 2 1 -- Colon
2 1 -- Lung 1 1 -- Endometrium 1 1 -- Cervix 1 0 -- Renal 1 1 --
TOTAL 27 12 44
TABLE-US-00010 TABLE 8 Other Tumors No. 8H9 reactive %
Hepatoblastoma 4 3 -- Wilm's tumor 8 7 -- Rhabdoid tumor 3 3 --
Paraganglioma 1 1 -- TOTAL 16 14 88
Heterogenous, non-specific cytoplasmic staining was noticed in
normal human pancreas, stomach, liver and adrenal cortex which was
diminished when 8H9F(ab')2 fragments were used instead of the whole
antibody for immunostaining. None of the other human tissues showed
reactivity with 8H9. In particular normal human brain tissue
sections including frontal lobe, spinal cord, pons and cerebellum
were completely negative. Normal tissues from cynomolgus monkey
also demonstrated similarly restricted reactivity with nonspecific
staining observed in stomach and liver (Table 4). The majority of
neuroectodermal and mesenchymal tumors tested showed positive
reactivity with 8H9, epithelial tumors to a lesser extent. 8H9
immunoreactivity was seen in a characteristic, homogenous, cell
membrane distribution in 272 of the 315 (86%) tumor samples
examined. 88% of neuroectodermal tumors, 95% of mesenchymal tumors
and 44% of epithelial tumors tested positive with 8H9 (Tables
4-8)
TABLE-US-00011 TABLE 4 Tissues Human Cynomolgous Frontal lobe
Negative Negative Pons Negative Negative Spinal cord Negative --
Cerebellum Negative Negative Lung Negative -- Heart Negative
Skeletal muscle Negative -- Thyroid Negative -- Testes Negative --
Pancreas cytoplasmic -- staining Adrenal cortex cytoplasmic
cytoplasmic staining staining Liver cytoplasmic cytoplasmic
staining staining Stomach -- Negative Sigmoid colon Negative --
Bone Marrow Negative -- Kidney Negative Negative
Indirect immunofluorescence 8H9 immunoreactivity in 34 NB,
melanoma, RMS, small cell lung cancer, OS, glioblastoma, leukemia,
breast cancer and colon cancer cell lines was tested using indirect
immunofluorescence. Moderate to strong cell membrane reactivity
with 8H9 was detected in 16/16 NB, 2/2 melanoma, 2/2 RMS, 1/1
glioblastoma multiforme, 3/3 breast cancer, and 1/1 colon cancer, 2
of 3 Ewing's/PNET, and 2 of the 3 OS cell lines. The small cell
lung cancer cell line HTB119 tested negative with 8H9 as did Jurkat
T-ALL cell line and EBV transformed lymphoblastoid cells. Normal
human bone marrow mononuclear cells (n=80) and hepatocytes (n=2)
had no reactivity with 8H9. Hepatocytes were isolated from human
cadavers and stained with 8H9. In contrast to anti-cytokeratin 18
and anti-HLA-class-1 antibodies which reacted strongly with surface
antigens, 8H9 staining was equivalent to control antibody.
Antigen modulation 8H9 binding to neuroblastoma line (NMB7),
rhabdomysarcoma (HTB82) and OS (U2OS) (measured by indirect
immunofluorescence) did not diminish significantly after 48 hr of
incubation at 37.degree. C. During the same period, binding to HLA
(MoAb HB95) diminished by 85% and to GD2 (3F8) by 55%, respectively
(FIG. 6). Electron microscopy using gold-labeled antibodies will be
more definitive in tracking antibody internalization, a process
clearly important for immunotoxins to be effective.
Enzyme-sensitivity There was a pronase dose-dependent reduction in
reactivity with 8H9 with 75-85% loss of immunofluorescence at a
final Pronase concentration of 0.3 mg/ml (FIG. 7). There was no
appreciable loss of reactivity with 3F8 (specific for the
ganglioside GD2) on NMB7 cells. Furthermore, the 8H9 antigen was
not sensitive to neuraminidase or phosphatidyl-inositol specific
phospholipase C (data not shown).
Biochemical Characterization of the novel antigen recognized by 8H9
Using a nonradioactive cell surface labeling technique, the antigen
was immunoprecipitated and analyzed on a SDS-PAGE..sup.101 In
brief, NB NMB7 or OS U2OS cells were biotinylated using
biotin-LC-NHS, lysed, precleared with protein-G sepharose, reacted
with antibody 8H9 and then immunoprecipitated in fresh protein G
sepharose. Antigen was then dissociated from the gel and separated
by SDS-PAGE. Following transblotting onto nitrocellulose membrane,
the protein bands were detected with HRP-strepavidin and visualized
by ECL. A band of 90 kDa under non-denaturing conditions and 96 kDa
in the presence of 2ME was found.
TABLE-US-00012 TABLE 9 % ID/gm % ID/gm TISSUE NB RMS TISSUE NB RMS
Time 24 h 172 h Time 24 h 172 h Tumor 8.3 5.3 Femur 0.7 0.3 Brain
0.2 0.1 Adrenal 1.0 0.3 Heart 2.1 0.8 Skin 0.2 0.4 Lung 0.8 1.4
Spine 1.7 0.4 Kidney 2.3 0.7 Blood 3.8 3.3 Liver 7.5 0.6 Spleen 6.7
0.6 Bladder 1.0 1.1 Stomach 0.3 0.5 Sm Intestine 0.3 0.3 Lg
Intestine 0.4 0.2 Muscle 0.2 0.2
Rat Anti-idiotypic MoAb specific for 8H9 By immunofluorescence the
antigen was sensitive to low temperatures. In view of the lability
of the antigen, we chose to synthesize anti-idiotypic antibodies as
surrogate antigen-mimics, in order to allow in vitro monitoring of
the antibody immunoreactivity e.g. after iodination of antibody
8H9. LOU/CN rats were immunized with protein-G purified 8H9
precipitated with goat-anti-mouse Ig, emulsified in CFA. Following
in vitro hybridization to the myelomas SP2/0 or 8653, 3 IgG2a
clones (2E9, 1E12, and 1F11) were selected for their high binding
and specificity. When tested against a panel of 23 other myelomas
or hybridoma antibodies, no cross-reactivity was found. The
anti-idiotypic hybridomas were cloned and antibodies produced by
high density miniPERM bioreactor from Unisyn Technologies
(Hopkinton, Mass.). The anti-idiotypic antibodies are further
purified by protein G (Pharmacia) affinity chromatography. To
further prove that these anti-idiotypic antibodies are
antigen-mimics, we immuno-enrich phagemids and screen scFv on solid
phase anti-idiotype, and successfully isolate a number of 8H9-scFv
with similar binding specificity to tumors as the parent 8H9 (see
below).
Tumor localization in xenografted SCID mice SCID mice with NB (NB)
xenografts were injected iv with 100 ug .sup.99mTc labeled 8H9.
Blood clearance was studied by blood cpm at various intervals after
injection. Mice were sacrificed at 24 hours and tissue uptake
expressed as percent injected dose per gram (Table 9). Significant
uptake in the reticulo-endothelial system in liver and spleen was
seen only with .sup.99mTc-8H9; none was evident when .sup.131I-3F8
was used. There was no significant difference between
.sup.99mTc-8H9 and .sup.131I-8H9 biodistribution. When the specific
activity of .sup.131I-8H9 was increased from 5 to >20 mCi/mg,
there was no degradation of tumor imaging or difference in
biodistribution. In SCID mice xenografted with RMS (RMS) xenograft,
following iv injection of 100 uCi of .sup.125I-8H9, selective tumor
uptake was evident at 4 to 172 hrs after injection, with a blood
T1/2 of 0.8 h and T1/2 of 26 h. Mean tumor/tissue ratios were
optimal at 172 h (for lung 4, kidney 7, liver 9, spleen 10, femur
16, muscle 21, brain 45). Average tumor/blood ratio were 0.7, 1.4
and 1.6, and tumor uptake was 9.5.+-.3.4, 13.3.+-.1.5, and
5.3.+-.0.9% injected dose per gm at 24, 48 and 172 h, respectively.
Control IgG1 MoAb antibody 2C9 remained in the blood pool without
localization to sc RMS xenografts. Tumor to normal tissue ratio was
favorable [range 5-55] for 8H9 (solid bar, FIG. 8) in contrast to
control MoAb 2C9.
8H9-ScFv We have synthesized single chain antibody (scFv) from 8H9.
Using polymerase chain reaction splicing by overlap extension,
variable regions of the heavy (V.sub.H) and light chains (V.sub.L)
of 8H9 were joined by a polylinker (L) (gly4Ser).sub.3 and selected
by phagemid expression. scFv was characterized by DNA sequencing,
western blots, in vitro ELISA, immunostaining/FACS, and idiotype
analysis. Using this scFv as a targeting unit, we are in the
process of synthesizing scFv-h 1-CH2-CH3 chimeric, scFv-m 3-CH2-CH3
chimeric, and T-bodies for retargeting T-cells.
Cell Populations Using 8H9-Magnetic Bead Immunoselection. ES is a
small round blue cell tumor of childhood characterized by a
t(11,22) in most patients. Because survival remains suboptimal with
standard therapy, many patients receive autologous stem cell
transplant and current trials investigating adoptive transfer of
autologous T cells in the context of immune therapy are underway.
However, approximately 50% of patients with advanced disease have
PCR detectable ES in peripheral blood and/or bone marrow and the
administration of autologous cell preparations contaminated with
tumor may contribute to disease relapse. To date, there is no
method reported for purging contaminated hematopoietic cell
populations or bone marrow preparations of ES. Merino et al in the
laboratory of Dr. Mackall at the Pediatric Oncology Branch, NCI,
Bethesda, Md., successfully optimized 8H9 for immunomagnetic
purging of ES. 8H9 bound to 9/9 of ES cell lines by flow cytometry.
Binding to peripheral blood mononuclear T cell and B cell
populations, as well as CD34+ cells from bone marrow was negative.
Utilizing immunomagnetic selection, 8H9 was used to isolate ES
cells from contaminated blood cell populations. Using real-time
quantitative nested PCR with the Lightcycler instrument, purging
efficiency was monitored by of t(11,22) RT-PCR. Contaminated
specimens were reacted with 8H9 and then incubated with rat
anti-mouse IgG1 magnetic beads. The sample was then run over a
Miltenyi Variomax negative selection column Recovery was
approximately 70% of the total PBMC. RNA was extracted from 10e7
cells from pre and post purge cell populations. Real time
quantitative PCR was performed with a level of sensitivity to one
tumor cell in 10e5 normal cells. A 2-log reduction of tumor cells
was achieved at a contamination of one tumor cell in 10 normal PBMC
and one tumor cell in 10e3 normal PBMC. Further studies evaluating
efficacy in clinical samples are underway. These results
demonstrate a potential new approach for purging contaminated
patient samples to be used in the context of autologous bone marrow
transplant and/or immunotherapy trials for ES.
8H9 purging of NB from marrow or blood cells In similar experiments
using Dynal beads coated with human anti-mouse IgG (Dynal, Lake
Success, N.Y.).sup.50 EGFP marked NMB7 cells could be
quantitatively removed in a one-cycle (either 8H9 or 3F8) or
2-cycle (8H9 followed by 3F8) immunomagnetic strategies (Table
10).
Research Design and Methods:
We will test if intravenous injections of iodine-131 labeled murine
MoAb 8H9 can detect primary and metastatic solid tumors. A total of
60 patients will be accrued over a period of 2 years.
#1: To define the level of agreement between .sup.131I-8H9 and
conventional imaging modalities in the detection of primary and
metastatic solid tumors in pediatrics.
1.1 Study Design
This is an open-label single arm study of .sup.131I-8H9, injected
intravenously at 10 mCi/1.73 m2 dose, after which patients will be
imaged at approximately day 0 to 1, 2 d, 3 d and whenever possible
6 to 7 d for dosimetry calculations. Blood samples will also be
obtained at least 12 times over the ensuing 7 days. Patients are
eligible for the protocol prior to their surgical resection or
biopsy of known or suspected tumor, or at the time of recurrent
tumor. .sup.131I-8H9 injection plus imaging can be repeated in each
patient up to a total of 3 times, but only if he/she has no HAMA
and no allergy to mouse proteins as evidenced by a negative skin
test.
1.2 Patient/Subject Inclusion Criteria
Gender and Minority Inclusion for Research Involving Human
Subjects:
Memorial Sloan Kettering Cancer Center has filed form HHS 441 (Re:
Civil Rights), form HHS 641 (Re: Handicapped individuals), and form
639-A (Re: sex discrimination). In selecting patients for study in
the proposed project, due notice is taken of the NIH Policy
concerning inclusion of women and minorities in clinical research
populations. The study population will be fully representative of
the whole range of patients seen at Memorial Hospital. No
exclusions will be made on the basis of gender or ethnicity.
However, because of the nature of these cancers which tend to
present in children and young adults, most the human subjects would
be of the younger age group.
Based on a December 1998-November 1999 analysis of the patient
population accrued to therapeutic clinical protocols, the racial
distribution of these patients were 16.6% black, Hispanic, or
Asian, 78.2% white and 5.2% other or unknown. The gender was 55.9%
male and 44.1% female. For the total patient population diagnosed
and treated at MSKCC in 1996, 26% were black, Hispanic, Asian or
Native American, 70% white and 6% unknown or not responding. Of
these patients, 38% were male and 62% female.
Participation of children: Children, adolescents and young adults
are the subjects of this clinical trial because of the nature of
these cancers. There is no age limit.
1.3.0 Evaluation During Treatment/Intervention
1.3.1 After injection of radiolabeled antibody, 1-2 cc of blood in
purple tops (EDTA) will be drawn at time 0, and around 15 min, 30
min, 1 h, 2 h, 4 h, 8 h, 18 h, 30 h, 42 h, 66 h, and once on day 6
or 7. Samples should be dated and timed. These samples are for
pharmacokinetic and for dosimetry studies. Patients with delayed
clearance will have one more imaging done between day 9 to 11.
TABLE-US-00013 Time Procedure day-10 start daily oral SSKI, cytomel
for thyroid blockade day 0 5 mCi of iodine-131 on 0.25 to 0.75 mg
of 8H9* blood samples at 0, and approximately 15 min, 30 min, 1 h,
2 h, 4 h, 8 h after injection day 0 Gamma camera scan plus whole
body counts day 1 Gamma camera scan plus whole body counts day 1
blood samples at approximately 18 h and 30 h day 2 Gamma camera
scan plus whole body counts day 2, 3 blood samples at approximately
42 h and 66 h day 5(or 6 or 7) Gamma camera scan plus whole body
counts and blood sample day 9 (or 10 or 11) if slow clearance day
14 Gamma camera scan plus whole body counts and blood sample Oral
SSKI and cytomel discontinued *Premedication with acetaminophen and
diphenhydramine.
1.3.2 Patients Will Undergo Gamma Imaging Days 0, 1, 2 and 5 or 6
or 7 after Injection. 1.3.3 Blood for HAMA q 1-2 Months 1.3.4
Tissue Biopsy is Recommended for Regions of Uptake by 8H9 Imaging
and Negative by Conventional Radiographic Techniques. 1.4.0
Biostatistics
To measure the level of agreement between conventional imaging
modality (CT, MRI, and nuclear scans) and antibody 8H9 imaging in
known and occult sites of disease. Index lesions will be confirmed
either by surgery or by disease-specific imaging (e.g. MIBG for
NB). For each individual, the proportion of sites found by 8H9
imaging will be scored. Given that there will be confirmation by
surgery or by disease-specific imaging, sensitivity analysis of 8H9
for each disease can be conducted. The probability of agreement or
positive predictive value will be calculated. The 95% confidence
intervals can be calculated within +/-31% for each disease (NB,
RMS, ES/PNET, DSRT, brain tumors and other sarcomas). The study
will be performed on a total of 60 patients (10 with NB, 10 RMS, 10
osteosacrcoma, 10 ES, 10 DSRT and 10 brain tumors plus other
8H9-positive tumors). Estimates on the level of agreement and the
level of tumor uptake will be computed separately in each disease
group. We are not using Kappa statistics for testing the
association between .sup.124I-3F8 imaging and other imaging
modalities (CT, MRI) since only patients with measurable or
evaluable tumors will be eligible for this protocol. In other
words, patients with no evidence of disease by conventional studies
will be not eligible. Therefore we cannot estimate the probability
of negative 8H9 imaging when conventional imaging studies are
negative, i.e. specificity analysis.
1.5.0 Preparation of .sup.131I-8H9
8H9 is produced under GMP conditions and packaged in glass vials.
.sup.131I is purchased from Amersham Inc. 8H9 will be labeled with
radioactive iodine using iodogen T method. The reaction mixture is
filtered through an ion exchange (AG1X8) filter (Biorad) to remove
free iodine. Protein incorporation is measured using TCA
precipitation or thin layer chromatography. Immunoreactivity is
measured by 2 separate methods (1) a solid phase microtiter
radioimmunoassay technique previously described,.sup.102 and (2)
anti-idiotype peak shift method, where anti-idiotypic antibody 2E9
is added at 50 to 1 molar ratio to .sup.131I-8H9 for 30 minutes on
ice with mixing. The percent cpm shifted on HPLC is a measure of
immunoreactivity. Radioiodinated 8H9 has a mean trichloroacetic
acid precipitability of >90%, and specific activity of
.sup.131I-8H9 averaging 10 mCi per mg protein. Administration of
.sup.131I-8H9 is undertaken within 1-2 hours of iodination to
reduce the possibility of radiolysis. Antibody radiolabeling is
carried out in the Central Isotope Laboratory under the supervision
of Dr. Ronald Finn, according to FDA guidelines on radiolabeled
biologics for human use.
1.6.0 Infusion of Radiolabeled Antibody Preparation and Monitoring
of Patient Response in Immediate Post-Infusion Period, Including
Radiation Safety Aftercare.
All radiolabeled MoAb preparations will be injected into patients
by a trained research nurse or physician. Strict observance of
appropriate radiation safety guidelines will be undertaken. The
procedure will be explained to the patient thoroughly prior to the
infusion by the physician, and appropriate pre-treatment (eg SSKI
drops, perchloracap) checked. The radiolabeled antibody will be
transported from the radiolabeling facility to the infusion area
loaded into the infusion delivery system by the physician. The
physician and nurse will be present throughout the infusion and in
the post-infusion period.
The infusion procedure will consist of the radiolabeled antibody
being administered intravenously either through a peripheral
intravenous catheter or an indwelling central catheter over a 20
minute period. All patients will have vital signs monitored prior
to and following the radiolabeled antibody infusion. Blood samples
for pharmacokinetic calculation will be obtained immediately
following the infusion, and at various time points thereafter as
outlined above. The patient will be seen by a physician daily while
hospitalized, and will be available for consultation (with
appropriate radiation safety personnel) with an oncologist or nurse
regarding issues relating to the radiolabeled antibody infusion or
radiation safety. The patient will also be imaged in the Nuclear
Medicine Department over the subsequent two week period, and all
imaging procedures performed will be supervised by the physician to
ensure that appropriate studies are obtained.
1.7.0 In Vitro Radioimmunoassay, ELISA, and Immunostaining
Quantitative in vitro assays on biologic fluids collected during
the course of clinical research studies in individual patients that
employ radiolabeled antibodies will be carried out. The methods
provided will include gamma counting of blood samples and HAMA
assays. HAMA titer in blood and serum will be correlated with the
clearance of .sup.131I-8H9
1.7.1 General counting procedures Aliquots of whole
blood/plasma/serum obtained from patients infused with radiolabeled
antibodies will be counted in a gamma counter with standards of
known activity for determination of sample activity. Tissue samples
obtained by biopsy or surgery will also be counted in a gamma
counter for determination of % injected dose/gram tissue.
Appropriate quality control procedures will be observed for
counting instruments and tissue specimens. 1.7.2 Quantitation of
HAMA by ELISA The presence of HAMA can modify the biodistribution
of .sup.131I-8H9. Although in naive patients HAMA is typically
undetectable, in patients with prior history of exposure to murine
antibodies or to 8H9, the presence of HAMA before and soon after
8H9 injection will need to be monitored. In addition, the formation
of HAMA was highly correlated with patient survival in the GD2-3F8
system, we plan to measure the serum antibody titer 6 months and 12
months after 8H9 exposure. The ELISA method has been described
previously..sup.11 Using F(ab').sub.2 fragments derived from the
three anti-idiotypic antibodies (2E9, 1E12, and 1F11), serum Ab3
will also be monitored as previously demonstrated for the GD2-3F8
system..sup.103,104 1.7.3 Quantitation of free circulating antigen
Since the biodistribution of 8H9 will be greatly affected by any
soluble antigen, patient sera before antibody injection will be
analyzed for antigenemia using an ELISA inhibition assay using a
modification of previously described method..sup.105 Microtiter
wells are coated with anti-idiotype MoAb 2E9. Serial serum
dilutions are used to inhibit the binding of biotinylated 8H9,
which can be detected by peroxidase-streptavidin. Upon washing,
color reaction is performed at room temperature using hydrogen
peroxide as substrate and o-phenylenediamine (Sigma, St. Louis,
Mo.) as chromogen. After stopping the reaction with 30 ul of 5N
sulfuric acid, optical density of the wells are then read using MRX
microplate reader (Dynex, Chantilly, Va.) and antibody titer
calculated in units/ml. 1.7.4 Immunostaining of tumor tissues Tumor
tissues will be tested for antigen expression using methods
previously described..sup.74
Anticipated results and potential pitfalls The injection of
.sup.131I-8H9 intravenously or intrathecally into cynomolgus
monkeys were well tolerated. Although we do not anticipate any
untoward side effects, patients will be closely monitored during
the antibody infusion with oxygen, antihistamines, epinephrine, and
hydrocortisone at the bed side. After the completion of antibody
injection, patients will be observed for at least 1 hour before
discharge from the clinic. Patients with unexpected grade 3-4
(other than urticaria, self-limited blood
pressure/pulse/temperature changes) or any life-threatening
toxicity will be reported immediately to the IRB and FDA. Given the
lability of the antigen in the cold (whether free or cell-bound),
immunoreactivity and soluble tumor antigen will be assayed using
the anti-idiotype as the antigen-mimic. The anti-idiotypic
antibodies are rat IgG1 MoAb purified by acid elution from protein
G affinity columns. They have remained stable despite acid
treatment, buffer changes and freezing and thawing. Soluble
antigens can interfere with tumor targeting. In vitro, patient
serum did not inhibit binding of 8H9 to its anti-idiotype. Indirect
immunofluorescence of a spectrum of cell lines showed persistence
of antigen and antibody on the cell surface at 37.degree. C. over
days. In xenograft biodistribution studies, there was no evidence
of antigen shedding that interferes with tumor imaging. Although
interference of 8H9 biodistribution by soluble antigen is unlikely,
we will document the absence by the ELISA inhibition assay. HAMA
response within the first two weeks after MoAb injection is rarely
observed among our patient population, partly because of the
intensity of the chemotherapy they received. However, some are
expected to mount a HAMA response when they are imaged a second
time. Clearly their HAMA will be monitored before and after
injection in order to interpret the biodistribution results.
Because of this sensitization, these patients may not be eligible
for subsequent MoAb therapies (as stated in the consent form).
However, we hypothesize that this HAMA response will help induce
the idiotype network, which may have benefit on patient survival,
analogous to our success with the murine 3F8-GD2 system we
described in preliminary results and progress report.
Interpretations and implications The ability of 8H9 to detect a
broad class of primary and metastatic solid tumors will be the
first step in defining the clinical utility of MoAb 8H9 in vivo.
Besides being a useful diagnostic tool, its therapeutic potential
will need to be explored. Clearly the amount of antibody deposited
in various organs need to be taken into account if these antibodies
are used to deliver radioisotopes, enzymes or drugs. Chimeric
antibodies with improved Fc effector functions and reduced
immunogenicity will also be explored. Immunocytokines and T-bodies
are also potential steps in future development of these agents.
#2: To Estimate the radiation dose per mCi of .sup.131I-8H9
delivered to tumors and to normal organs in patients.
To obtain data necessary for patient dosimetry, patients will be
injected, intravenously, with .sup.131I-8H9 according to their
surface area, i.e. 10 mCi/1.73 m2. A total of three or four gamma
camera images will be obtained within a 1 to 2 week period
following injection. The following schedule is recommended but may
be altered, if necessary: 1-4 h after injection (day 0) and then
again on days 2, day 3, and day 6 or 7. If warranted, due to slow
clearance kinetics, imaging on days 9, 10 or 11 may also be
performed. Using this schedule weekend imaging may be avoided
regardless of the weekday injected. Scan types and imaging
parameters are listed below:
2.1 Data Collection:
SPOT and SPECT images will be collected over pre-selected "index"
tumor lesions, as identified from previously obtained CT or MR
images.
2.1.1 Blood collection Blood samples will be collected as follows:
prior to injection, and at 0, 15, 30 min, then 1 h, 2 h, 4 h, 8 h,
18 h or 30 h, 42 h, 66 h, day 6 or 7 following the injection.
Plasma or serum will be collected and counted from each sample and
the results will be expressed as per cent of the injected
radioactivity per L serum or blood volume.
TABLE-US-00014 static spot view (SPOT) HEHR collimation 10 to 20
min acquisition time dual-window acquisition for scatter correction
128 .times. 128 .times. 16 matrix size
TABLE-US-00015 SPECT HEHR collimation 6 degrees or 64 views in stop
and shoot, elliptical orbit mode 1 to 4 min/view (0.5 to 2 h
acquisition time on a dual-headed camera) dual-window acquisition
for scatter correction 64 .times. 64 .times. 16 matrix size
TABLE-US-00016 whole-body sweep (SWEEP) high-energy,
high-resolution (HEHR) collimation, 8 to 12 cm/min sweep speed (20
to 25 min acquisition time) dual-window acquisition for scatter
correction 256 .times. 1024 .times. 16 matrix size
TABLE-US-00017 Imaging schedule: Imaging day SWEEP SPOT SPECT 0 X X
1, 2 X X X 5, 6 or 7 X X 9, 10, or 11 X X
2.1.2 Pharmacokinetics Modeling Blood time-activity curves from
serial blood samples and from ROI's around sequential SPECT images
of the heart (when available). This data will be fitted, together
with the whole-body clearance kinetics, to a pharmacokinetic model
of antibody distribution. Previously developed models have been
used for this type of analysis, further details regarding the
approach have been published..sup.106 2.1.3 Patient-specific
dosimetry (3D-ID) The pharmacokinetic data obtained from SPECT and
planar imaging and blood sampling will be combined with anatomical
imaging information (MR or CT) to estimate the absorbed dose to
tumor and selected normal organs that would be expected from a
therapeutic injection of .sup.131I-8H9. The methodology for this
has been previously described..sup.107-115 2.2 Tumor volume
determinations Tumor volumes will be determined from CT or MRI when
available. Patients with known disease at other sites are imaged in
additional areas. All CT images will be transferred for display in
3D-ID; images collected at MSKCC will be transferred digitally,
film from other institutions will be scanned using a Lumisys
digital film scanner. Using 3D-ID, the consulting radiologist will
review the images with the research technician. The research
technician will then draw contours around the tumor regions; the
contours will be reviewed by the consulting radiologist and
adjusted, as needed. In some cases, disease may be represented by a
collection of very small positive nodes; in those cases a contour
around the group will be drawn and used in the volume assessment.
Volume determination using 3D-ID is performed by summing the areas
of regions that have been defined by the user on all slices making
up the tumor. This general approach has been previously validated
for CT. Although potentially labor-intensive, such a tumor
outline-specific method is significantly more accurate than
techniques based upon greater and minor diameters (i.e.,
ellipsoidal models)). The errors associated with CT-based volume
estimation and the factors influencing these errors have been
examined and will be considered in the volume determinations
described above. A reliable total-body tumor burden will not be
achievable for all patients, either because of the small volume of
disease, or for cases in which lesions detected by SPECT are not
visible by CT. 2.3 Red marrow dosimetry Bone marrow dosimetry will
be performed according to the recommended guidelines, described in
the AAPM recommendations,.sup.116 i.e. blood time-activity curves
will be multiplied by the appropriate factor (0.2-0.4) to derive
marrow time-activity curves and absorbed dose to red marrow.
S-Factors provided in MIRDOSE 3 will be used for the calculations.
This data will be compared with direct measurement of the marrow
activity from ROI's drawn over marrow cavities on SPECT images. The
quantitative capability of SPECT will allow us to verify the
accuracy of bone marrow dosimetry determined from activity levels,
and the rate of antibody clearance from marrow, from the standard
analysis of serial blood samples. 2.4 Three-dimensional dosimetry
To perform 3D dosimetry, it is first necessary to register a set of
nuclear medicine images (SPECT), depicting the radiolabeled
antibody distribution to an anatomical imaging modality (CT or
MRI). We have extensive experience with the clinical implementation
of the Pelizzari and Chen method..sup.117 This technique requires
that the user delineate the same surface on both sets of imaging
modalities. When necessary, a SPECT transmission study is performed
to obtain the appropriate surface. The program attempts to maximize
the correlation of a set of several hundred points on the surface
as identified on one scan (the "hat"), with a solid model of the
same surface derived from the other scan (the "head"). A non-linear
least-squares search is used to minimize the sum of the squares of
distances from each "hat" point to the nearest point on the "head"
surface. The coordinates of the "hat" are translated, rotated and
scaled to provide the best fit. Users may control which parameters
are varied during the search. The final set of transformations are
then used to convert the coordinates of one image into those of the
other. Phantom studies indicate that the Pelizzari and Chen
technique for registration of SPECT to CT is accurate to within 3
mm. The Nuclear Medicine Service at MSKCC has performed such
registration for over 100 patient studies. The Pellizari and Chen
package has also been used for thoracic and abdominal study
registration by Chen and his collaborators at the University of
Chicago (personal communication). Both the Chicago group and us
have also included contours for liver and/or spleen along with the
body contours. This further improves registration by providing more
contours for the minimization algorithm. In some cases, a
radioactive band has also been used as an aid to
registration..sup.117 We are currently comparing this method with
alternative algorithms for image registration for the whole
images..sup.117-120
Correlated serial SPECT images can be used to determine cumulative
activity distributions by fitting and integrating an exponential
uptake and/or clearance to the specific activity within an ROI over
the tumor or organ. The variation in activity within individual
voxels can be taken into account, through a weighted sum of the
counts/activity within the corresponding voxel over time. Given
such a distribution of the cumulated activity, a software package,
3D-ID, has been developed, to calculate the dose distribution.
Target contours are drawn on side-by-side enlarged SPECT and CT/MR
image slices that are selected from a scrollable image display.
Contours drawn in one modality simultaneously appear in the other.
The user may switch between modalities by positioning the cursor in
the appropriate window. This provides for the simultaneous use of
both imaging modalities to define tumor (e.g. using SPECT) and
normal organ (using CT/MR) contours. The dose to all voxels within
the target volume is obtained by convolving the activity
distribution with a point kernel table of absorbed dose versus
distance. Patient-specific S-factors may be calculated by defining
source organ contours and assigning unit activity to all voxels
within each source. The "dose" to a given target is thus the
patient-specific S-factor. Dose histograms and patient-specific
organ and tumor S-factors generated using 3D-ID in combination with
SPECT will provide important information in understanding tumor
response and organ toxicity in radioimmunotherapy.
Photon dose kernels for 14 radionuclides of interest in internal
emitter therapy have been recently published..sup.112. Explicit
expressions of radionuclide photon dose kernels, necessary for
three-dimensional dosimetry, were not previously available. We
recently described the overall structure and methodologies of a
software package for three-dimensional internal dosimetry (3D-ID)
calculations..sup.107,113 A series of software modules that address
the logistical issues of performing patient-specific
three-dimensional dosimetry were detailed. Software tools have been
developed to combine images from different modalities, define
regions-of-interest using available multi-modality data and
identify source and target volumes for dosimetry. A point-kernel
based dosimetry calculation has been implemented and several
different approaches for displaying the spatial distribution of
absorbed dose in a biologically pertinent manner were also
described. The dose calculation, itself, was carried out in a
separate module, so that different calculation schemes including
Monte Carlo, may be used with 3D-ID.
2.5 Anticipated Results
The major sources of error in carrying out absorbed dose
calculations are: 1. Inaccuracies in imaging-derived activity
concentration estimates. 2. Mismatch between standard anatomy (used
for dosimetry calculations) and individual patient anatomy. 3.
Assumption of uniformity in the spatial distribution of
radioactivity on both a micro (mm to mm) and macro (cm) scale. When
applying conventional (MIRD Committee) approaches to estimating
absorbed dose it is understood that the estimate is derived from a
model which includes a certain number of assumptions. This approach
has been sufficient in estimating doses for diagnostic applications
wherein typical doses are already far below toxicity. An objective
of radioimmunotherapy, however, is to treat to normal organ
tolerance. In such a scenario, accurate, patient-specific dosimetry
is critical. The dosimetry methodologies that will be used in this
proposal address point 2 and a portion of point 3; dose
calculations are performed for individual patient geometries and
the spatial distribution of radioactivity in tumor or normal organs
is accounted for on a macroscopic (cm) scale. In the past using
planar imaging kinetics to project the kinetics of the spatial
distribution had additional pitfalls. Although SPECT-based activity
determinations are a step forward, we expect these inaccuracies in
imaging derived activity to be further reduced when I-124-8H9
Positron emission tomography is used. This is an area of active
development at Memorial Sloan Kettering in the last
decade..sup.121
Conventional dosimetry yields estimates of the absorbed dose,
averaged over a normal organ or tumor volume. The methodology
implemented in this proposal will yield the spatial distribution of
absorbed dose as isodose contours, overlayed upon a 3-D CT image
set. This makes it possible to evaluate the anatomical distribution
of absorbed dose to tissues and from this, assess the potential
impact in terms of toxicity. For example, the dose to surrounding
tissue from activity that has concentrated in a tumor contained
within a normal organ can be obtained by this means.
2.6 Interpretations and Implications
The average absorbed dose to a tumor may not reflect potential
therapeutic efficacy and tumor shrinkage. That portion of a tumor
volume receiving the lowest absorbed dose will lead to treatment
failure regardless of the dose delivered to other regions of the
tumor volume. The 3D-ID software package provides detailed
information regarding the spatial distribution of absorbed dose
within a target volume. This information is depicted as dose-volume
histograms, wherein the fraction of tumor volume receiving a
particular absorbed dose is plotted against absorbed dose. Using
such information it will be possible to better assess the
likelihood of tumor control. For example, if the average dose over
a tumor volume is 2 to 3 Gy and a small region within this volume
receives only 0.1 Gy, then treatment will be unsuccessful.
REFERENCES
1. DiMaggio J J, Scheinberg D A, Houghton A N: Monoclonal antibody
therapy of cancer. In: Pinedo H M, Chabner B A, Longo D L, (eds.):
Cancer Chemotherapy and Biological Response Modifiers, Annual 11,
Elsevier Science Publishers B.V., (Biomedical Division), 1990, pp
177-203 2. Schlom J: Monoclonal Antibodies in cancer therapy: Basic
principles. In: DeVita V T, Hellman S, Rosenberg S A, (eds.):
Biologic therapy of cancer, 2nd ed. Philadelphia, J.B. Lippincott
Co, 1995, pp 507-520 3. Koehler G, Milstein C: Continuous culture
of fused cells secreting antibody of pre-defined specificity.
Nature 256:495-496, 1975 4. Moffat R, Pinsky C M, Hammershaimb L,
et al: Clinical utility of external immunoscintigraphy with the
IMMU-4 technetium-99m Fab' antibody fragment in patients undergoing
surgery for carcinoma of the colon and rectum: results of a
pivotal, phase III trial. The Immunomedics Study Group. J Clin
Oncol 14(8):2295-2305, 1996 5. Maloney D G, Grillo-Lopez A J,
Bodkin D J, et al: IDEC-C2B8: Results of a phase I multiple-dose
trial in patients with relapsed non-hodgkin's lymphoma. J Clin
Oncol 15:3266-3274, 1997 6. Cobleigh M A, Vogel C L, Tripathy D, et
al: Multinational study of the efficacy and safety of humanized
anti-HER2 monoclonal antibody in women who have HER2-overexpressing
metastatic breast cancer that has progressed after chemotherapy for
metastatic disease. J Clin Oncol 17:2639-2648, 1999 7. Bigner D D,
Brown M T, Friedman A H: Iodine-131-labeled antitenascin monoclonal
antibody 8106 treatment of patients with recurrent malignant
gliomas: phase I trial results. Journal Clinical Oncology
16:2202-2212, 1998 8. Jurcic J G, Caron P C, Miller W H: Sequential
targeted therapy for acute promyelocytic leukemia with all-trans
retinoic acid and anti-CD33 monoclonal antibody M195. Leuk
9:244-248, 1995 9. Meredith R F, Khazaeli M B, Plott W E: Phase II
study of dual 131I-labeled monoclonal antibody therapy with
interferon in patients with metastatic colorectal cancer. Clin Can
Res 2:1811-1818, 1996 10. Yeh S D, Larson S M, Burch L, et al:
Radioimmunodetection of neuroblastoma with iodine-131-3F8:
Correlation with biopsy, iodine-131-Metaiodobenzylguanidine (MIBG)
and standard diagnostic modalities. J Nucl Med 32:769-776, 1991 11.
Cheung N K V, Kushner B H, Cheung I Y, et al: Anti-GD2 antibody
treatment of minimal residual stage 4 neuroblastoma diagnosed at
more than 1 year of age. J Clin Oncol 16:3053-3060, 1998 12.
Wheldon T E, O'Donoghue J A, Barrett A, Michalowski A S: The
curability of tumors of differing size by targeted radiotherapy
using 131-1 or 90-Y. Radiother Oncol 21:91-99, 1991 13. Wilder R B,
DeNardo G L, DeNardo S J: Radioimmunotherapy: recent results and
future directions. J Clin Oncol 14:1383-1400, 1996 14. Zalutsky M
R, McLendon R E, Garg P K, et al: Radioimmunotherapy of neoplastic
meningitis in rats using an alpha-particle-emitting
immunoconjugate. Cancer Res 54:4719-4725, 1994 15. McDevitt M R,
Sgouros G, Finn R D, et al: Radioimmunotherapy with alpha-emitting
nuclides. Eur J Nucl Med 25:1341-1351, 1998 16. Lode H N, Xiang R,
Becker J C, et al: Immunocytokines: A promising approach to cancer
immunotherapy. Pharmacology Therapeutics 80:277-292, 1998 17.
DeNardo S J, DeNardo G L, DeNardo D G, et al: Antibody phage
libraries for the next generation of tumor targeting
radioimmunotherapeutics. Clin Can Res 5:3213s-3218s, 1999 18.
DeNardo S J, DeNardo G L, Brush J, Carter P: Phage Library-derived
human anti-TETA anti anti-DOTA ScFv for pretargeting RIT. Hybridoma
18:13-21, 1999 19. Eshhar Z, Waks T, Gross G, Schindler D G:
Specific activation and targeting of cytotoxic lymphocytes through
chimeric single chains consisting of antibody-binding domains and
the or zeta subunits of the immunoglobulin and T-cell receptors.
Proc Natl Acad Sci USA 90:720-24, 1993 20. Altenschmidt U, Kahl R,
Moritz D, et al: Cytolysis of tumor cells expressing the
Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted
naive T lymphocytes. Clin Can Res 2:1001-1008, 1996 21. Krause A,
Guo H F, Tan C, et al: Antigen-dependent CD-28 signaling enhances
survival and proliferation in genetically modified activated human
primary T lymphocytes. Exp Med 188:619-626, 1998 22. Garin-Chesa P,
Fellinger E J, Huvos A G: Immunohistochemical analysis of neural
cell adhesion molecules. Am J Pathol 139:275-286, 1991 23. Ritter
G, Livingston P O: Ganglioside antigens expressed by human cancer
cells. Semin Cancer Biol 2:401-409, 1991 24. Wikstrand C J, Longee
D C, McLendon R E, et al: Lactotetraose series ganglioside
3',6'-isoLD1 in tumors of central nervous and other systems in
vitro and in vivo. Cancer Res 53:120-126, 1993 25. Seeger R C,
Danon Y L, Rayner S A, Hoover F: Definition of a Thy-1 determinant
on human neuroblastoma, glioma, sarcoma, and teratoma cells with a
monoclonal antibody. J Immunol 128:983-989, 1982 26. Ylagan, Quinn
L R: B: CD44 expression in astrocytic tumors. Modern Pathology
10:1239-1246, 1997 27. Kaaijp P, Troost D, Morsink F, et al:
Expression of CD44 splice variants in human primary brain tumors.
Journal of Neuro-Oncology 26:185-190, 1995 28. Richardson R B,
Davies A G, Bourne S P, et al: Radioimmunolocalization of human
brain tumors. Biodistribution of radiolabelled monoclonal antibody
UJ13A. Eur J Nucl Med 12:313-320, 1986 29. Papanastassiou V, Pizer
B L, Coakham H B, et al: Treatment of recurrent and cystic
malignant gliomas by a single intracavitary injection of
131I-monoclonal antibody: Feasibility, pharmacokinetics and
dosimetry. Br J Cancer 67:144-151, 1993 30. Kishima H, Shimizu K,
Tamura K, et al: Monoclonal antibody ONS-21 recognizes integrin a3
in gliomas and gliomas and medulloblastomas. Br J Cancer
79:333-339, 1998 31. Moriuchi S, Shimuzu K, Miyao Y, Hayakawa T:
Characterization of a new mouse monoclonal antibody (ONS-M21)
reactive with both medulloblastomas and gliomas. Br J Cancer
68:831-837, 1993 32. Erikson H P, Lighter V A: Hexabrachion protein
(tenascin, cytotactin, brachionectin) in connective tissues,
embryonic tissues and tumors. Adv Cell Biol 2:55-90, 1988 33. Riva
P, Frnceschi G, Frattarelli M, et al: 131I radioconjugated
antibodies for the locoregional radioimmunotherapy of high-grade
malignant glioma--phase I and II study. Acta Oncol 38:351-359, 1999
34. Kuan C T, Reist C J, Foulon C F, et al: 125I-labeled
anti-epidermal growth factor receptor vIII single-chain Fv exhibits
specific and high-level targeting of glioma xenografts. Clin Can
Res 5:1539-1549, 1999 35. Wikstrand C J, Hale L P, Batra S K, et
al: Monoclonal Antibodies against EGFRvIII are Tumor Specific and
React with Breast and Lung Carcinomas and Malignant Gliomas. Cancer
Res 55:3140-48, 1995 36. Kondo S, Miyatake S, Iwasaki K, et al:
Human glioma-specific antigens detected by monoclonal antibodies.
Neurosurgery 30:506-511, 1992 37. Dastidar S G, Sharma S K:
Monoclonal antibody against human glioblastoma multiforme (U-87Mg)
immunoprecipitates a protein of monoclonal mass 38 KDa and inhibits
tumor growth in nude mice. J Neuroimmuno 56:91-98, 1995 38. Mihara
Y, Matsukado Y, Goto S, et al: Monoclonal antibody against
ependymoma-derived cell line. Journal of Neuro-Oncology 12:1-11,
1992 39. Wang N P, Marx J, McNutt M A: Expression of myogenic
regulatory proteins (myogenin and MyoD1) in small blue round cell
tumors of childhood. Am J Pathol 147:1799-1810, 1995 40. Weidner N,
Tjoe J. Immunohistochemical profile of monoclonal antibody O13 that
recognizes glycoprotein 930/32MIC2 and is useful in diagnosing
ewing's sarcoma and peripheral neuroepithelioma. American Journal
of Surgical Pathology 18:486-494, 1994 41. Weidner N, Tjoe J.
Immunohistochemical profile of monoclonal antibody O13 that
recognizes glycoprotein 930/32MIC2 and is useful in diagnosing
ewing's sarcoma and peripheral neuroepithelioma. Am J Pathol
18:486-494, 1994 42. Heiner J, Miraldi F D, Kallick S, et al: In
vivo targeting of GD2 specific monoclonal antibody in human
osteogenic sarcoma xenografts. Cancer Res 47:5377-5381, 1987 43.
Price M R, Campbell D G, Robyn R A: Characteristics of the cell
surface antigen p72, associated with a variety of human tumors and
mitogen-stimulated T-lymphoblasts. FEBS Letters 171:31-35, 1984 44.
Spendlove I, James L L, Carmichael J, Durrant L G: Decay
accelerating factor (CD55): a target for cancer vaccines? Cancer
Res 59:2282-2286, 1999 45. Gorlick R, Huvos A G, Heller G, et al:
Expression of HER2/erbB-2 correlates with survival in osteosarcoma.
J Clin Oncol 17:2781-2788, 1999 46. Bruland O, Fodstad O, Funderud
S, Pihl A: New monoclonal antibodies specific for human sarcomas.
Int J Cancer 15:27-31, 1986 47. Cheung N K, Saarinen U M, Neely J
E, et al: Monoclonal antibodies to a glycolipid antigen on human
neuroblastoma cells. Cancer Res 45:2642-2649, 1985 48. Modak S,
Gultekin S H, Kramer K, et al: Novel tumor-associated surface
antigen: broad distribution among neuroectodermal, mesenchymal and
epithelial tumors, with restricted distribution in normal tissues.
Proceedings of ASCO 17:449a, 1998 49. Cheung N K, Heller G, Kushner
B H, et al: Detection of metastatic neuroblastoma in bone marrow:
when is routine marrow histology insensitive? J Clin Oncol
15:2807-2817, 1997 50. Ghossein R A, Osman I, Bhattacharya S, et
al: Detection of circulating prostatic tumor cells using immunobead
reverse transcriptase polymerase chain reaction for prostatic
specific membrane antigen mRNA. Diag Mol Path 8:59-65, 1999 51.
Leung W, Chen A R, Klann R C, et al: Frequent detection of tumor
cells in hematopoietic grafts in neuroblastoma and ewing's sarcoma.
Bone Marrow Transpl 22:971-979, 1998 52. Mueller B M, Romerdahl C
A, Gillies S D, Reisfeld R A: Enhancement of antibody-dependent
cytotoxicity with a chimeric anti-GD2 antibody. J Immunol
144:1382-1386, 1990 53. Santos A D, Kashmiri V S, Horan P H, et al:
Generation and characterization of a single gene-encoded
single-chain-tetravalent antitumor antibody. Clin Can Res
5:3118s-3123s, 1999 54. Guo H F, Rivlin K, Dubel S, Cheung N K V:
Recombinant anti-ganglioside GD2 scFv-streptavidin fusion protein
for tumor pretargeting. Proc Am Assoc Cancer Res 37:469, 1996
(abstract) 55. Fagnou C, Michon J, Peter M, et al: Presence of
tumor cells in bone marrow but not in blood is associated with
adverse prognosis in patients with ewing's tumor. J Clin Oncol
16:1707-1711, 1998 56. Cheung N K, Kushner B H, LaQuaglia M,
Lindsley K: Treatment of advanced stage neuroblastoma. In: Reghavan
D, Scher H I, Leibel S A, Lange P, (eds.): Principles and Practice
of Genitourinary Oncology. Philadelphia, J.B. Lippincott Company,
1997, pp 1101-1111 57. Brodeur G M, Castleberry R P: Neuroblastoma.
In: Pizzo P A, Poplack D G, (eds.): Principles and Practice of
Pediatric Oncology, 3rd ed. Philadelphia, J.B. Lippincott Company,
1997, pp 761-797 chapter 29 58. Cheung N K V: Biological and
molecular approaches to diagnosis and treatment. section I.
Principles of Immunotherapy. In: Pizzo P A, Poplack D G, (eds.):
Principles and Practice of Pediatric Oncology, 3rd ed. ed.
Philadelphia, J.B. Lippincott Company, 1997, pp 323-342 59. Larson
S M, Sgouros G, Cheung N K: Antibodies in cancer therapy:
Radioisotope conjugates. In: DeVita V T, Hellman S, Rosenberg S A,
(eds.): Biologic Therapy of Cancer, 2nd ed. Philadelphia, J.B.
Lippincott Co., 1995, pp 534-552 60. Levy R, Miller R A: Antibodies
in cancer therapy: B-cell lymphomas. In: DeVita V T, Hellman S,
Rosenberg S A, (eds.): Biologic therapy of cancer, 1st ed.
Philadelphia, J.B. Lippincott Co, 1991, pp 512-522 61. Reisfeld R
A, Mueller B M, Handgretinger R: Potential of genetically
engineered anti-ganglioside GD2 antibodies for cancer
immunotherapy. In: Progress in Brain Search (Svennerhol, L, Asbury,
A K, Reisfeld, R A, Sandhoff, K, Suzuki, K, Tettamani, G, Toffano,
G, vol. 101. Cambridge, UK, Elsevier Trends Journals, 1994, pp
201-212 62. Munn D H, Cheung N K: Interleukin-2 enhancement of
monoclonal antibody-mediated cellular cytotoxicity (ADCC) against
human melanoma. Cancer Res 47:6600-6605, 1987 63. Hank J A,
Robinson R R, Surfus J, et al: Augmentation of antibody dependent
cell mediated cytotoxicity following in vivo therapy with
recombinant interleukin-2. Cancer Res 50:5234-5239, 1990 64.
Kushner B H, Cheung N K: GM-CSF enhances 3F8 monoclonal
antibody-dependent cellular cytotoxicity against human melanoma and
neuroblastoma. Blood 73:1936-1941, 1989 65. Kushner B H, Cheung N K
V: Absolute requirement of CD11/CD18 adhesion molecules, FcRII and
phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated
neutrophil anti-human tumor cytotoxicity. Blood 79:1484-1490, 1992
66. Cheung N K V, Walter E I, Smith-Mensah W H, et al:
Decay-accelerating factor protects human tumor cells from
complement-mediated cytotoxicity in vitro. J Clin Invest
81:1122-1128, 1988 67. Saarinen U M, Coccia P F, Gerson S L, et al:
Eradication of neuroblastoma cells in vitro by monoclonal antibody
and human complement: method for purging autologous bone marrow.
Cancer Res 45:5969-5975, 1985 68. Munn D H, Cheung N K:
Antibody-dependent antitumor cytotoxicity by human monocytes
cultured with recombinant macrophage colony-stimulating factor.
Induction of efficient antibody-mediated antitumor cytotoxicity not
detected by isotope release assays. Exp Med 170:511-526, 1989 69.
Munn D H, Cheung N K: Phagocytosis of tumor cells by human
monocytes cultured in recombinant macrophage colony-stimulating
factor. J Exp Med 172:231-237, 1990 70. Sabzevari H, Gillies S D,
Mueller B M, et al: A recombinant antibody-interleukin 2 fusion
protein suppresses growth of hepatic human neuroblastoma metastases
in severe combined immunodeficiency mice. Proceeds of the National
Academy of Science USA 91:9626-9630, 1994 71. Murray J L,
Cunningham J E, Brewer H M, et al: Phase I trial of murine
anti-ganglioside (GD2) monoclonal antibody (Mab) 14G2A in cancer
patients. J Biol Resp Modif 1991 (Soc. Biol. Therapy Meeting
Abstract 1991.) 72. Ugur O, Kostakoglu L, Hui E T, et al:
Comparison of the targeting characteristics of various
radioimmunoconjugates for radioimmunotherapy of neuroblastoma:
Dosimetry calculations incorporating cross-organ beta doses. Nucl
Med Biol 23:1-8, 1996 73. Mujoo K, Reisfeld R A, Cheung L,
Rosenblum M G: A potent and specific immunotoxin for tumor cells
expressing disialoganglioside GD2. Cancer Immunol Immunother
34:198-204, 1991 74. Gottstein C, Schon G, Tawadros S, et al:
Antidisialoganglioside Ricin A-chain immunotoxins show potent
anti-tumor effects in vitro and in a disseminated human
neuroblastoma severe combined immunodeficiency mouse model. Cancer
Res 54:6186-6193, 1994 75. Holzer U, Bethge W, Krull F, et al:
Superantigen-staphylococcal-enterotoxin-A-dependent and
antibody-targeted lysis of GD2-positive neruoblastoma cells. Cancer
Immunol Immunother 41:129-136, 1995 76. Cheung N K, Lazarus H,
Miraldi F D, et al: Ganglioside GD2 specific monoclonal antibody
3F8--a phase I study in patients with neuroblastoma and malignant
melanoma. J Clin Oncol 5:1430-1440, 1987 77. Cheung N K, Lazarus H,
Miraldi F D, et al: Reassessment of patient response to monoclonal
antibody 3F8. J Clin Oncol 10:671-672, 1992 78. Murray J L,
Cunningham J E, Brewer H, et al: Phase I trial of murine monoclonal
antibody 14G2a administered by prolonged intravenous infusion in
patients with neuroectodermal tumors. J Clin Oncol 12:184-193, 1994
79. Saleh M N, Khazaeli M B, Wheeler R H, et al: Phase I trial of
the chimeric anti-GD2 monoclonal antibody ch 14.18 in patients with
malignant melanoma. Human Antibodies Hybridomas 3:19-24, 1992 80.
Handgretinger R, Anderson K, Lang P, et al: A phase I study of
human/mouse chimeric antiganglioside GD2 antibody ch 14.18 in
patients with neuroblastoma. Eur J Cancer 31:261-267, 1995 81.
Uttenreuther-Fischer M M, Huang C-S, Reisfeld R A, Yu A L:
Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in phase 1 trial
in pediatric cancer patients. Cancer Immunol Immunother 41:29-36,
1995 82. Cheung N K V, Kushner B H, Yeh S J, Larson S M: 3F8
monoclonal antibody treatment of patients with stage 1V
neuroblastoma: a phase II study. In: Evans A E, Guillio J D,
Biedler J L, et al, (eds.): Advances in Neuroblastoma Research,
vol. 4. New York, Wiley Liss, 1994, pp 319-329 83. Yu A L, Gillies
S D, Reisfeld R A: Phase I clinical trial of ch14.18 in patients
with refractory neuroblastoma. Proc Am Soc Clin Oncol 10:318, 1991
84. Handgretinger R, Baader P, Dopfer R, et al: A phase I study of
neuroblastoma with the anti-ganglioside GD2 antibody 14.G2a. Cancer
Immunol Immunother 35:199-204, 1992 85. Cheung N K: Biological and
Molecular Approaches to Treatment. Immunotherapy. In: Pizzo P A,
Poplack D G, (eds.): Principles and Practice of Pediatric Oncology,
2nd ed. Philadelphia, J.B. Lippincott Company, 1992, pp 357-370 86.
Miraldi F D, Nelson A D, Kraly C, et al: Diagnostic imaging of
human neuroblastoma with radiolabeled antibody. Radiology
161:413-418, 1986 87. Arbit E, Yeh S J, Cheung N K, Larson S M:
Quantitative Immunoimaging of gliomas in humans with
anti-ganglioside monoclonal antibodies. J Neurosurg 76:399a, 1991
88. Daghighian F, Pentlow K S, Larson S M, et al: Development of a
method to measure kinetics of radiolabeled monoclonal antibody in
human tumors with applications to microdosimetry: Positron emission
tomography studies of iodine-124 labeled 3F8 monoclonal antibody in
glioma. Eur J Nucl Med 20:402-409, 1993 89. Grant S C, Kostacoglu
L, Kris M G, et al:
Radioimmunodetection of small-cell lung cancer using the anti-GD2
ganglioside monoclonal antibody 3F8: a pilot trial. Eur J Nucl Med
23:145-149, 1996 90. Cheung N K V, Kushner B H, Yeh S J, Larson S
M: 3F8 monoclonal antibody treatment of patients with stage 1V
neuroblastoma: A phase II Study. Int J Oncol 12:1299-1306, 1998 91.
Cheung N K, Yeh S D, Kushner B H, et al: Phase I study of
radioimmunotherapy of neuroblastoma using iodine 131 labeled 3F8.
In: Prog. Clin. Biol. Res: Advances in Neuroblastoma Research 4.
New York, Wiley Liss, 1994, pp 329 92. Larson S M, Pentlow K S,
Volkow N D, et al: PET scanning of iodine-124-3F8 as an approach to
tumor dosimetry during treatment planning for radioimmunotherapy in
a child with neuroblastoma. J Nucl Med 33:2020-2023, 1992 93.
Pentlow K S, Graham M C, Lambrecht R M, et al: Quantitative imaging
of 1-124 using positron emission tomography with applications to
radioimmunodiagnosis and radioimmunotherapy. Medical Physics
18:357-366, 1991 94. Pentlow K S, Graham M C, Lambrecht R M:
Quantitative imaging of iodine-124 with PET. J Nucl Med
37:1557-1562, 1996 95. Lewellen T K, Kohlmyer S G, Miyaoka R S,
Kaplan M S: Investigation of the performance of the general
electric advance positron emission tomograph in 3D mode.
Transplantation Nuclear Science 1996 96. Kramer K, Cheung N K V,
DiResta G, et al: Pharmacokinetics and acute toxicology of
intraventricular I-monoclonal antibody targeting disialoganglioside
in non-human primates. J Neuro Oncol 1996 97. Dropcho E J, Saleh M
N, Grizzle W E, Oh S J: Peripheral neuropathy following treatment
of melanoma with a murine anti-GD2 monoclonal antibody (MoAb).
Neurology 1992 (abstract in press) 98. Saleh M N, Khazaeli M B,
Wheeler R H, et al: A phase I trial of the murine monoclonal
anti-GD2 antibody 14.G2a in metastatic melanoma. Cancer Res
52:4342-4347, 1992 99. Saleh M N, Wheeler R H, Khazaeli M B, et al:
A phase I trial of chimeric anti-GD2 monoclonal antibody C14.18 in
patients with metastatic melanoma. International Conference on
Monoclonal Antibody Immunoconjugates for cancer 1991 (abstract)
100. Cheung N K, Cheung I Y, Canete A, et al: Antibody response to
murine anti-GD2 monoclonal antibodies: Correlation with patient
survival. Cancer Res 54:2228-2233, 1994 101. Drengler R L, Kuhn J
G, Schaaf L J, et al: Phase I and pharmacokinetic trial of oral
irinotecan administered daily for 5 days every 3 weeks in patients
with solid tumors. J Clin Oncol 17:685-696, 1999 102. Cheung N K,
Landmeier B, Neely J, et al: Complete tumor ablation with iodine
131-radiolabeled disialoganglioside GD2 specific monoclonal
antibody against human neuroblastoma xenografted in nude mice. J
Natl Cancer Inst 77:739-745, 1986 103. Cheung I Y, Cheung N K V,
Kushner B H: Induction of Ab3' following anti-GD2 monoclonal
antibody 3F8 therapy predicts survival among patients (pts) with
advanced neuroblastoma. Proc Am Assoc Cancer Res 40:574, 1999 104.
Chen S, Caragine T, Cheung N K, Tomlinson S: Surface antigen
expression and complement susceptibility of differentiated
neuroblastoma clones. Am J Pathol In press: 1999 105. Cheung N K,
Canete A, Cheung I Y, et al: Disialoganglioside GD2 anti-idiotypic
monoclonal antibodies. Int J Cancer 54:499-505, 1993 106. Loh A,
Sgouros G, O'Donoghue J A, et al: A pharmacokinetic model of
1311-G250 antibody in patients with renal cell carcinoma. J Nucl
Med 3:484-489, 1998 107. Kolbert K S, Sgouros G, Scott A M, et al:
Implementation and evaluation of patient-specific three dimensional
internal dosimetry. J Nucl Med 38:301-308, 1997 108. Sgouros G,
Jureidini 1M, Scott A M, et al: Bone marrow dosimetry: Regional
variability of marrow-localizing antibody. J Nucl Med 37:695-698,
1996 109. Sgouros G, Divgi C R, Scott A M, et al: Hematologic
toxicity in radioimmunotherapy: An evaluation of different
predictive measures. J Nucl Med 37:43P-44P, 1996 110. Sgouros G,
Deland D, Loh A C, et al: Marrow and whole-body absorbed dose vs
marrow toxicity following 131I-G250 antibody therapy in patients
with renal-cell carcinoma. J Nucl Med 38:252P, 1997 111. Sgouros G:
Treatment planning for internal emitter therapy: methods,
applications and clinical implications. 1996 112. Furhang E E,
Sgouros G, Chui C S: Radionuclide photon dose kernels for internal
emitter dosimetry. Medical Physics 23:759-764, 1996 113. Furhang E
E, Chui C S, Sgouros G: A monte carlo approach to patient-specific
dosimetry. Medical Physics 23:1523-1529, 1996 114. Furhang E E,
Chui C S, Kolbert K S, et al: Implementation of a monte carlo
dosimetry method for patient-specific internal emitter therapy.
Medical Physics 24:1163-1172, 1997 115. Sgouros G: Yttrium-90
biodistribution by yttrium-87 imaging: a feasibility analysis.
Medical Physics 2000 116. Siegel J A, Wessels B W, Watson E E, et
al: Bone marrow dosimetry and toxicity in radioimmunotherapy.
Antibody Immunoconjugates Radiopharmaceuticals 3:213-233, 1990 117.
Scott A M, Macapinlac H, Zhang J, et al: Image registration of
SPECT and CT images using an external fiduciary band and
three-dimensional surface fitting in metastatic thyroid cancer. J
Nucl Med 36:100-103, 1995 118. Woods R P, Mazziotta J C, Cherry S
R: Quantification of brain function. Tracer kinetics and image
analysis in brain PET. 1993 (ED. Uemura K, Elseiver Science
Publishers) 119. Talairach J, Tournouz P: Co-planar stereotactic
atlas of the human brain. Georg Thieme Verlag 1988 120. Meyer C R,
Boes J L, Kim B, et al: Demonstration of accuracy and clinical
versatility of mutual information for automatic multimodality image
fusion using affine and thin-plate spline warped geometric
deformations. Medical Image Analysis 1:195-206, 1997 121. Sgouros
G, Chiu S, Pentlow K S, et al: Three-dimensional dosimetry for
radioimmunotherapy treatment planning J Nucl Med 34:1595-1601,
1993
Third Series of Experiments
Immunomagnetic Purging of Ewing's Sarcoma from Blood: Quantitation
by Real-Time PCR
Ewing's sarcoma is a childhood tumor characterized by a t(11,22) in
most patients. Because survival remains suboptimal with standard
therapy, many patients receive autologous stem cell transplant and
trials investigating adoptive transfer of autologous T cells in the
context of immune therapy are underway. However, approximately 50%
of patients with advanced disease have PCR detectable disease in
peripheral blood and/or bone marrow and administration of
contaminated autologous cell preparations may contribute to disease
relapse. To date, there is no reported method for purging
contaminated hematopoietic cell populations of Ewing's Sarcoma. 8H9
is a mouse monoclonal IgG1 antibody previously reported to react
with 21/21 Ewing's sarcoma/PNET tumors (Proc ASCO 17:44a, 1998).
Peripheral blood T cell and B cell populations and CD34+ cells from
bone marrow analyzed by flow cytometry for binding of 8H9 were
negative. We sought to use magnetic bead immunoselection of 8H9
labeled cells to purge peripheral blood cell populations
contaminated with Ewing's sarcoma. Using real-time quantitative
nested PCR with Lightcycler, we monitored purging efficiency by
evaluation of t(11,22) by RT-PCR. Contaminated specimens were
labeled with 8H9 and incubated with rat anti-mouse IgG1 magnetic
beads. The sample was then run over a Miltenyi Variomax negative
selection column. Recovery was approximately 70%. RNA was extracted
from 10e7 cells from pre and post purge cell populations. Real-time
quantitative PCR was performed with a level of sensitivity to one
tumor cell in 10e5 normal cells. We demonstrated at least a two-log
reduction of tumor in preparations contaminated at a ratio of 1:10
normal PBMC and 1:10e3 normal PBMC. Further studies evaluating
efficacy in clinical samples are underway. These results
demonstrate a potential new approach for purging contaminated
patient samples to be used in the context of autologous bone marrow
transplant and/or immunotherapy trials for Ewing's sarcoma.
Immunomagnetic Purging of Ewing's Sarcoma from Blood and Bone
Marrow: Quantitation by Real-Time PCR
The propensity for hematogenous spread of Ewing's sarcoma and the
resulting contamination of autologous cell preparations complicates
the use of cellular therapies in this disease. To date, there has
been no reported method for purging marrow and other cellular
products of Ewing's sarcoma. In this paper, we introduce monoclonal
antibody 8H9, which showed binding by flow cytometry to 9/9 Ewing's
sarcoma cell lines studied. Binding to lymphocytes and bone marrow
progenitor cells was negative. In order to test whether 8H9 could
be used for immunomagnetic based purging, normal PBMCs or bone
marrow cells were artificially contaminated with varying amounts of
Ewing's sarcoma. Quantitative PCR or t(11;22) was shown to
accurately measure the level of contamination with a sensitivity of
1:10.sup.6. Samples were then purged using the Miltenyi Variomax
negative selection system selecting for monoclonal antibody 8H9
bound cells. A 2 to 3-log reduction in tumor burden was
consistently observed following immunomagnetic selection. In
clinical non-mobilized apheresis studied, Ewing's contamination
ranged between 1:10.sup.5-1:10.sup.6. Therefore 8H9 based purging
of clinical samples is predicted to result in a contamination level
which is below the limit of detection by sensitive quantitative
PCR. These results demonstrate a potential new approach for purging
contaminated patient samples to be used in the context of
autologous bone marrow transplant and/or immunotherapy trials for
Ewing's sarcoma.
Current concepts hold that Ewing's sarcoma is a systemic disease
from the time of onset as demonstrated by the observation that over
90% of patients with clinically localized disease will recur
distantly if treated with local measures alone [Jaffe, 1976 #49].
Indeed, the generally accepted factor responsible for the recent
improvement in survival observed in patients with clinically
localized disease is control of hematogenously disseminated
micrometastasis via neoadjuvant multi-agent chemotherapy.sup.1.
Recently, the use of sensitive molecular monitoring to detect
circulating Ewing's sarcoma cells has confirmed hematogenous
dissemination in a substantial number of patients with Ewing's
sarcoma. West et al .sup.2 found a 25% incidence of translocation
(11;22) positivity in the peripheral blood or bone marrow in
patients with clinically localized disease, and higher rates have
been observed in other series .sup.3 and in patients with overt
metastatic disease. .sup.3, 4 Interestingly, in the reports by de
Alava and Toretsky, evidence for positivity in peripheral blood
persisted following initiation of chemotherapy suggesting that
ongoing dissemination may occur intermittently throughout treatment
protocols.
In an attempt to improve survival in high-risk patients with
Ewing's sarcoma, several groups have studied the use of high dose
chemotherapy followed by bone marrow or peripheral stem cell
transplantation. .sup.5-17. Up to a 40% survival in poor risk
patients has been reported after high dose therapy followed by
autologous stem cells in contrast to historical survival rates of
0-20% with chemotherapy/radiation therapy alone .sup.5, 6. One
factor complicating the use of autologous stem cell products in
therapy of Ewing's sarcoma is the propensity for hematogenous
dissemination with resultant contamination of stem cell products.
In one report, despite CD34 based positive selection for progenitor
cells, autologous peripheral blood progenitor preparations were
shown to contain EWS/FLI1 translocation positive cells in 54% of
samples evaluated .sup.4. While the true clinical impact of
contaminating tumor cells in autologous products remains unclear,
genetically marked tumor cells residing in autologous bone marrow
have been shown to be present at disease relapse in patients with
neuroblastoma and AML.sup.18, 19. Similar concern regarding the
potential for autologous cell preparations to contribute to disease
recurrence arise in the context of immune based therapy trials
which are currently being undertaken and involve the transfer of
autologous T cells harvested prior to the initiation of
therapy.sup.20.
To date there has been no method reported for purging autologous
hematopoietic cells of Ewing's sarcoma. In this report, we
introduce a monoclonal antibody based purging technique which
allows us to reduce the tumor burden in contaminated bone marrow or
peripheral blood specimens by two to three logs which is predicted
to be below the limit of detection of PCR positivity in the vast
majority of clinically contaminated specimens.
Materials and Methods
Monoclonal Antibody Production (Memorial Sloan-Kettering Cancer
Center)
Cell Preparations
Peripheral Blood Mononuclear Cells: PBMCs used in tumor spiking
experiments were obtained by ficoll-based density gradient
separation of the fresh buffy coat fraction of normal healthy donor
blood units obtained at the Department of Transfusion Medicine,
Clinical Center, NCI according to approved protocols. For analysis
of T cell reactivity to anti-CD3 monoclonal antibody following
purging, PBMCs were T cell enriched using a negative selection
column (R & D Biosystems, Minneapolis) which results in a
purity of approximately 80%. Patient apheresis samples analyzed for
contamination were obtained as part of NCI POB 97-0052 following
informed consent. Leukopheresis procedures were done using the
CS3000 Plus (Fenwal Division, Baxter, Deerfield, Ill.) which
processed 5-15 liters of blood volume. Countercurrent centrifugal
elutriation of the apheresis product was performed using a Beckman
J-6M centrifuge equipped with a JE 5.0 rotor (Beckman Instruments,
Palo Alto, Calif.) in HBSS without magnesium, calcium and phenol
red (BioWhittaker, Walkersville, Md.) at a centrifuge speed of 3000
rpm (1725.times.g).sup.21. Cell fractions (450-550 ml each) were
collected at flow rates of 120, 140, and 190 ml/min during
centrifugation and at 190 ml/min with the rotor off (RO). The first
two fractions are typically enriched for lymphocytes while the last
two fractions are enriched for monocytes. All fractions were
cryopreserved in 10% DMSO (Cryoserv, Research Industries, Salt Lake
City, Utah), RPMI with penicillin, streptomycin and L-glutamine and
25% fetal calf serum.
Progenitor Cells: CD34+ cells used for purging experiments were
selected using the Miltenyi Variomax.RTM. direct isolation system
(Miltenyi, Auburn, Calif.) from cryopreserved peripheral stem cells
from a Ewing's sarcoma patient obtained for therapeutic use at
Children's National Medical Center, Washington, D.C. according to
approved protocols and following informed consent. Stem cells were
used for research purposes after the patient's death. These cells
were not positive by RT-PCR for Ewing's sarcoma and were therefore
artificially contaminated for the purging experiments. Non-CD34
selected bone marrow used for purging experiments and enriched
CD34+ populations used in the CFU assay were obtained from fresh
human marrow harvested from normal volunteers according to approved
protocols and following informed consent (Poietics Laboratories,
Gaithersburg, Md.). The mononuclear fraction was obtained by
ficoll-based density gradient separation, and subsequently enriched
for CD 34+ cells by the Miltenyi Variomax.RTM. (Miltenyi, Auburn,
Calif.) direct CD34 selection system.
Tumor Cell Lines: Ewing's sarcoma cell lines used for screening
included TC71, 5838, RD-ES, CHP100, A4573 which have been
previously reported .sup.22 and JR and SB which are cell lines
derived from patients treated at the National Cancer Institute
which have also been previously reported .sup.22. LG was a cell
line derived from a patient with isolated intrarenal recurrence of
Ewing's sarcoma treated with resection at the University of
Maryland.
Flow Cytometry Analysis
Flow cytometric analysis was performed using the Becton-Dickinson
FacsCalibur machine. Briefly, fluorescence data were collected
using a 3-decade log amplification on 10,000 viable gated cells as
determined by forward and side light scatter intensity. Monoclonal
antibodies used for immunofluorescence were: MoAb 8H9, murine IgG1
isotype, goat anti-mouse IgG1-FITC, CD3-PE (S4.1), CD34-PE (581)
Caltag (Burlingame, Calif.), CD99-FITC (TU12) (Pharmingen, San
Diego, Calif.). For immunofluorescence analysis, cells were
incubated with antibody at a concentration of 1 ug/10.sup.6 cells
for 20 minutes at 4.degree., followed by washing with PBS with 0.2%
human serum albumin and 0.1% Sodium Azide. For 8H9 and isotype
staining, this was followed by incubation with goat anti-mouse FITC
for 10 minutes at 4.degree. C. followed by washing prior to
analysis.
Immunomagnetic Purging
All cell products were spiked with tumor cells from the Ewing's
sarcoma cell line TC71 at the levels of contamination indicated for
individual experiments. For purging of CD34+ peripheral stem cells,
a total of 10.times.10.sup.6 were spiked. 1.times.10.sup.6 cells
were analyzed for pre-purged and post-purged PCR. For PBMC and
non-CD34 selected bone marrow specimens, 30-80.times.10.sup.6 cells
were spiked with TC71 with 10.times.10.sup.6 cells analyzed for
pre-purged and post-purged PCR. For purging, cells were incubated
at 4.degree. C. with monoclonal antibody 8H9 at a concentration of
1 ug/10.sup.6 total cells for 20 minutes and washed with buffer
(PBS, 0.5% BSA, 2 mM EDTA). Cells were then incubated with rat
anti-mouse IgG1 magnetic beads (Miltenyi, Auburn, Calif.) at a
ratio of 1:1 for 20 minutes at 4.degree. C. Purging was
accomplished using the Miltenyi Variomax.RTM. system wherein the
sample is run over the Miltenyi (Auburn, Calif.) AS depletion
column with a flower resistor of 24G. Cells from the depleted
fraction were then washed with 3 cc buffer. The positively selected
fractions of cells was removed by releasing the column from the
magnet and washing with 3 cc buffer, and analyzed by PCR where
indicated. In cases where clonogenicity of the positive fraction
was evaluated, the positive fraction was pelleted and resuspended
in RPMI with 10% FCS, L-glutamine (4 uM), penicillin (100 u/ml),
and streptomycin (100 ug/ml), and placed in an incubator at
37.degree. C. with 5% CO.sub.2 for 5 days.
Conventional PCR
For analysis of contamination of patient apheresis fractions, RNA
was extracted from 20-50.times.10.sup.6 cells using TRIzol Reagent
(Life Technologies, Rockville, Md.) or guanidinium
isothiocynate/CsCl method .sup.23. After cDNA was generated from
250 ng RNA using a random hexamer, PCR was performed with Perkin
Elmer GeneAmp PCR system 2400 using ESPB1 and ESBP2 primers and the
following conditions: 40 cycles 95.degree. C. 30 s, 60.degree. C.
30 s, 72.degree. C. 30 s followed by 72.degree. C. for 7 minutes.
To assess the integrity and quantity RNA, a PCR reaction with GAPDH
primers was performed for each patient sample. 10 ul of each PCR
product were run on 1.3% TBE agarose gel and transferred to a nylon
membrane. A [.sup.32P].gamma.-ATP 20-mer oligonucleotide probe was
generated using T4 polynucleotide kinase. The membrane was
hybridized using ExpressHyb Hybridization Solution (Clontech, Palo
Alto, Calif.) according to the manufacturer's instructions. The
membrane was then exposed to Kodak Xomat film (Kodak, Rochester,
N.Y.) for 24-144 hours.
Real-Time Quantitative PCR
Real-time quantitative PCR was performed using the Lightcycler.RTM.
Instrument (Roche Molecular Biochemicals, Indianapolis, Ind.). RNA
was extracted from 10.times.10.sup.6 cells from all samples except
for the CD34+ population in which 1.times.10.sup.6 cells were used.
The Trizol.RTM. phenol/chloroform extraction or RNA-easy columns
(Qiagen, Valencia, Calif.) were used. The 1.sup.st Strand Synthesis
kit (Roche, Indianapolis, Ind.) was used to generate cDNA from 1 ug
of RNA from each sample. PCR was then run on 5 ul of cDNA on the
Lightcycler.RTM. instrument with primers ESBP1 and ESBP2 for 40
cycles. In cases where nested PCR was performed, an initial 20
cycles of PCR were carried out with the primer pair ESBPI-ESBP2
followed by 40 additional cycles using 2 ul of the product of the
first reaction using the primer pair EWS 696-Fl1 1041 By
conventional PCR, primer pair ESBP1-ESBP2, and EWS 696-FLI 1041
generate fragments of 310 bp and 205 bp respectively. Both sets of
primers are outside the breakpoint of the EWS/FLI 1 translocation.
In the initial evaluation of the quantitative PCR, both nested and
non-nested Lightcycler.RTM. PCR products were confirmed by size
using 1% TAE agarose gel with ethidium bromide (data not shown).
Hybridization probes spanning the EWS/FLI 1 breakpoint were used to
detect target template in the Lightcyler reaction. To provide a
positive control and to quantitate total amplified RNA, G6PD was
amplified from 5 ul of cDNA and analyzed using sequence specific
hybridization probes G6PDHP1 and G6PDHP2. On all hybridization
probes, the 5' probe (HP1) was labeled at the 3' end with
Fluorescein, the 3' probe (HP2) was labeled at the 5' end with
Lightcycler Red 640 and phosphorylated at the 3' end. Cycle
crossing number was ascertained at the point in which all samples
had entered the log linear phase. Cycle crossing number was used to
determine log cell concentration according to a standard curve. The
standard curve was generated by amplifying 5 ul of cDNA derived
from 1 ug of RNA from 10.times.10.sup.6 normal PBMCs spiked with
TC71 tumor cells at decreasing concentrations from 1:10 to
1:10.sup.7.
TABLE-US-00018 Sequences [.sup.32P].gamma.Probe
5'TACTCTCAGCAGAACACCTATG SEQ ID NO: 4 Primers ESBP1 5' CGA CTA GTT
ATG ATC AGA GCA 3' SEQ ID NO: 5 ESBP2 5' CCG TTG CTC TGT ATT CTT
ACT GA 3' SEQ ID NO: 6 EWS 696 5' AGC AGC TAT GGA CAG CAG 3' SEQ ID
NO: 7 FLI 1 1041 5' TTG AGG CCA GAA TTC ATG TT 3' SEQ ID NO: 8
G6PD1 5' CCG GAT CGA CCA CTA CCT GGG CAA G 3' SEQ ID NO: 9 G6PD 2
5' GTT CCC CAC GTA CTG GCC CAG GAC SEQ ID NO: 10 CA 3' Lightcycler
Hybridization Probes EWSHP1 5' TAT AGC CAA CAG AGC AGC AGC TAC-F 3'
SEQ ID NO: 11 EWSHP2 5' LC RED 640-GGC AGC AGA ACC CTT SEQ ID NO:
12 CTT-P 3' G6PDHP1 5' GTT CCA GAT GGG GCC GAA GAT CCT SEQ ID NO:
13 GTT G-F 3' G6PDHP2 5' LC RED 640-CAA ATC TCA GCA CCA TGA SEQ ID
NO: 14 GGT TCT GCA C-P 3'
OKT3 Mediated Proliferation of Purged T Cell Specimens
1.times.10.sup.8 CD3 enriched cells were contaminated with Ewing's
sarcoma at a level of 1:10.sup.3. Cells from pre-purged and
post-purged samples were added in triplicate to a 96 well plate at
a concentration of 2.times.10.sup.5 cells/well containing
decreasing concentrations of plate bound anti-CD3 antibody OKT3
(Ortho Biotech Inc., Raritan, N.J.) from 100 ug/ml to 3 ug/ml.
Cells were incubated with 200 ul of RPMI with 10% FCS, L-glutamine,
penicillin, and streptomycin for a 48 hours and then pulsed with 1
uCi of [.sup.3H] thymidine per well. Cells were harvested after 18
hours of pulsing and .sup.3H incorporation was enumerated using the
TopCount NXT (Packard, Meriden Conn.). Subtracting background
activity with media alone generated net counts.
CFU Assay
CD34+ cells were enriched from pre- and post-purged samples from
fresh human bone marrow using the Miltenyi.RTM. direct CD34+
progenitor isolation kit. 35.times.10.sup.6 bone marrow mononuclear
cells from each sample were run over a positive selection (MS)
column yielding a CD34+ enriched population with estimated purities
of >70% .sup.24. 1000 cells were plated in triplicate in
methylcellulose media supplemented with recombinant cytokines
(MethoCultGF+H4435, Stem cell Technologies, Vancouver, BC). CFUs
were counted after 14 days of culture.
Results
Monoclonal Antibody 8H9 Binds all Ewing's Sarcoma Cell Lines Tested
but not Normal Lymphocytes or Hematopoietic Progenitors.
In order to identify a potential reagent that could be used to
target contaminating Ewing's sarcoma cells, monoclonal antibodies
induced via immunization with neuroblastoma were screened for cross
reactivity with Ewing's sarcoma. Monoclonal antibody 8H9 was
observed to bind to 9/9 Ewing's sarcoma cell lines evaluated (FIG.
9). The level of reactivity was variable with some lines showing
diminished levels of reactivity compared to CD99 whereas two lines
(SB and RD-ES), showed increased reactivity compared to CD99
Importantly, lymphoid and hematopoietic populations showed no
reactivity with 8H9 as shown in FIG. 10a (CD3 gated PBMC), and FIG.
10b (CD34 gated bone marrow cells), whereas CD99 showed significant
binding to T cell populations.
Quantification of Ewing's Sarcoma Contamination Using Real-Time PCR
of Artificially Contaminated Specimens Accurately Quantitates Tumor
Contamination with Sensitivity to 1:10.sup.6.
To study whether immunomagnetic purging of marrow and peripheral
blood populations contaminated with Ewing's sarcoma could be
quantitatively monitored, we sought to devise an approach wherein
variable levels of contamination could be quantified using RT-PCR.
We began by artificially contaminating PBMC populations with a log
based titration of Ewing's contamination (e.g. 1:10-1:10.sup.7).
The degree of contamination was evaluated using real-time PCR.
Using a non-nested PCR, we observed linear relationships across
four log levels of contamination, (FIG. 11a). However, the limit of
detection for a non-nested PCR was 1 tumor cell in 10.sup.4
background cells. In an effort to increase the sensitivity, we also
evaluated nested PCR, using an initial 20 cycles of amplification
followed by 40 cycles amplification with internal primers. With
this approach, quantitative accuracy was lost for only the highest
level of contamination, which likely began to plateau with the
initial 20 cycles (11b). However, quantitative accuracy was
observed for levels of contamination between 1:100 to 1:10.sup.6
was observed (FIG. 11c). Because 10.times.10.sup.6 starting cells
were used in these experiments, we can estimate that using the
nested approach, amplification was accomplished from 10
contaminating cells. This confirmed the utility of quantitative PCR
to provide an accurate quantitative assessment of tumor
contamination with a level of sensitivity of one tumor in 10.sup.6
background cells, thus allowing measurements of the efficacy of 8H9
based approaches for purging of Ewing's sarcoma cells.
MoAb 8H9 Based Immunomagnetic Purging Yields a 2 to 3-Log Reduction
in Artificially Contaminated Peripheral Blood and Bone Marrow
Populations.
In order to purge hematopoietic progenitor populations of Ewing's
sarcoma, variably contaminated 8H9 incubated bone marrow or
peripheral blood stem cell populations were run over a
Variomax.RTM. negative selection column as described in methods.
Non-nested PCR evaluation of non-CD34 selected bone marrow from a
healthy donor spiked with Ewing's sarcoma cells at a level of 1:100
is shown in FIG. 12a. These results demonstrate a 2-log reduction
in tumor following 8H9 based purging. To evaluate the efficiency of
8H9 based purging with progenitor contamination at lower levels and
to assess the ability to purge CD34+ selected cells, CD34+ selected
cells from G-CSF mobilized peripheral blood were spiked at a level
of 1:10.sup.3 and purged as shown in FIG. 12b. Using the
quantitative PCR, we observed a 3-log reduction in the level of
contamination following one run over the column.
In the next experiments, evaluation of the ability to purge
contaminated PBMC populations was undertaken. Similar to the
results observed with CD34+ enriched peripheral blood stem cells,
at least a 3-log reduction in contamination following 8H9 based
purging of PBMCs contaminated at 1:100 was attained as shown in
FIG. 12c. Evaluation of purging of PBMCs contaminated at a lower
level (1:10.sup.3) is shown in FIG. 12d where a 3-log reduction is
again observed. In each of these experiments analysis of the
positive fraction demonstrated PCR positivity confirming selection
of contaminating Ewing's cells (data not shown). To account for any
variation from the expected uniform amounts of starting RNA or
cDNA, G6PD amplification was performed from each sample in a
quantitative fashion. We observed a variation in crossing time
(reflective of starting template) of less than 2% in all of the
samples indicating a low degree of variation in starting template
between samples and confirming viable RNA and cDNA in the negative
samples (data not shown). These results suggested that monoclonal
antibody 8H9 may be a suitable candidate for immunomagnetic based
purging of contaminated blood, bone marrow, and CD 34+ enriched
progenitor populations specimens with the likelihood for purging to
PCR negativity being high if the level of contamination present in
clinical samples is less than 1:10.sup.4.
Contamination of Non-Mobilized Patient Apheresis Fractions with
Ewing's Sarcoma is Between 1:10.sup.5-1:10.sup.6.
In order to evaluate the degree of contamination typically observed
in clinical specimens, we studied non-mobilized peripheral blood
apheresis specimens derived from patients treated on immunotherapy
trials for Ewing's sarcoma at our institution. We observed a 66%
(8/12) incidence of t(11,22) PCR positivity in non-mobilized
apheresis specimens acquired for use in immunotherapy protocols as
analyzed by conventional PCR (Table 1). As shown in Table 1, all
elutriated apheresis fractions were observed to contain tumor with
variability across individual patients. When elutriated apheresis
specimens from several patients at presentation of metastatic
Ewing's sarcoma were analyzed using quantitative PCR, this level of
contamination was estimated to be between 1:10.sup.5 and 1:10.sup.6
with similar levels of contamination sometimes observed in multiple
apheresis fractions. (FIG. 13). Patient A (top panel) showed
positivity of all fractions at levels of approximately 1:10.sup.6.
Patient B (middle panel) showed a level of contamination of
approximately 1:10.sup.6 in the 120 ml/min (lymphocyte) fraction
with no evidence for positivity in the 190 ml/min or rotor off
(monocyte) fractions. Patient C (bottom panel) showed a level of
contamination between 1:10.sup.5 and 1:10.sup.6 in multiple
fractions. In no instance have we observed levels of contamination
greater than 1:10.sup.4. Therefore, because clinical specimens
contaminated with Ewing's sarcoma appears to be in the range of
1:10.sup.5-1:10.sup.6, it is anticipated that reduction in
contamination to at least 1:10.sup.7 following 8H9 based purging
will be achievable in the vast majority of patients.
TABLE-US-00019 TABLE 1 Contamination of non-mobilized apheresis
fractions with Ewing's sarcoma as analyzed by conventional PCR.
Lymphocyte Fractions Monocyte Fractions Patient Number 120 ml/min
140 ml/min 190 ml/min Rotor Off 1 N/A Positive Negative Positive 2
Positive Positive Positive Positive 3 Positive Negative Positive
N/A 4 Negative Negative N/A Positive 5 Negative Negative Negative
Negative 6 N/A Negative Negative Positive 7 Negative Negative
Negative Negative 8 Negative Negative Negative Negative 9 Negative
Positive Positive Negative 10 Positive Positive N/A Positive 11
Negative Negative Negative Negative 12 Negative Negative Negative
Positive Positive indicates band hybridized with the EWS/FLI
radiolabeled probe. Negative indicated no band was noted. N/A
indicated that no RNA was obtained for that fraction.
8H9 Based Purging does not Adversely Affect Stem Cell or T Cell
Function.
To further evaluate the clinical feasibility of this technique for
purging of bone marrow or PBSC autografts, we sought to confirm
retained proliferative and differentiating capacity in 8H9 purged
bone marrow populations. We studied CFU formation following purging
as an assay of CD34 function. We compared CFU formation before and
after purging in CD34 selected bone marrow cells cultured in
methylcellulose media with recombinant cytokines before and after
purging (FIG. 14). We observed normal colony numbers and morphology
in both samples with no significant difference between samples
indicating that CD34+ progenitors remain functional following 8H9
based purging.
T Cell Proliferation is Unchanged Before and after Purging.
Because T cells can contribute to post chemotherapy immune
reconstitution.sup.25, we are currently utilizing autologous T cell
infusions harvested prior to initiation of chemotherapy in order to
study effects on immune reconstitution. In order to study T cell
function following 8H9 based purging, we evaluated T cell
proliferation following anti-CD3 cross linking as a measure of T
cell function. We compared T cell proliferation unmanipulated T
cells and 8H9 based purged T cells. As shown in FIG. 15, there was
no difference in T cell proliferation elicited by plate bound OKT3
antibody at concentrations ranging from 100 ug/ml to 3 ug/nl as
measured by [.sup.3H] thymidine uptake indicating that T cell
proliferative capacity is retained following 8H9 based purging
(FIG. 15).
Discussion
The contribution of contaminated autologous preparations to disease
relapse following autologous SCT in solid tumor patients is not
fully known. Rill and Brenner et al. have shown than in certain
solid tumors, tumors contaminating autologous grafts are
tumorigenic and present at relapse.sup.18, 19. In a disease such as
Ewing's sarcoma, which has been shown to have a high degree of
hematogenous spread, this becomes an important issue in the context
of therapies which utilize autologous cells. In high-risk patients,
survival after high dose chemotherapy followed by stem cell rescue
continues to be suboptimal with the most common cause of death due
to disease relapse. Contamination of autografts with subsequent
survival and clonogenic growth of tumor post-infusion cannot be
excluded as contributing to this poor prognosis. In addition to the
medical consequences of the administration of contaminated products
to patients, psychologically there is reluctance on the part of
patients and their families to receive contaminated products. It
follows, therefore, that if a purging method was available, its
evaluation for use in patients receiving autologous products is
warranted.
An ideal purging method should target only tumor cells and show no
binding to normal cell populations. The identification of such a
tumor specific antigen has historically posed a challenge in
Ewing's sarcoma. While CD99 typically shows high expression on
Ewing's sarcoma cells, it is also expressed on T cells (FIG. 10a)
and CD 34 stem cells.sup.26, making it unsuitable for purging
hematologic products. Monoclonal antibody 8H9 was initially
developed due to its reactivity with neuroblastoma and was
subsequently reported to react with 19/19 fresh Ewing's
sarcoma/PNET tumor confirming that 8H9 reactivity is not limited to
established cell lines. .sup.27. Our results (FIG. 9) confirmed
this reactivity in all Ewing's cell lines evaluated. Since this
antibody showed no reactivity with T cells and CD34+ cells, it was
ideally suited for purging. Indeed, we demonstrated a 2-3 log
reduction in all experiments following one run over the negative
selection column. In the clinical setting of autologous stem cell
transplant, the combination of positive selection for CD34+ cells,
which results in an approximate 2-log passive depletion of tumor
.sup.28, 29, followed by 8H9 purging of tumor cells would be
expected to result in up to 5 logs of depletion, which is predicted
to be well below the limit of detection using currently available
techniques. Further, even in the setting of autologous T cell
transplantation, as potentially used in the context of immune
reconstitutive therapies.sup.20, the use of 8H9 based purging with
its 2-3 log reduction will substantially diminish the tumor burden
contained in autologous cellular products.
This is the first published report of 8H9 as a Ewing's reactive
monoclonal antibody. Interestingly, 8H9 also shows reactivity with
several rhabdomyosarcoma and osteosarcoma cell lines (data not
shown). This introduces the exciting possibility of a sarcoma
specific antibody with potential applications in immune directed
therapy. In addition, identification and characterization of the
tumor specific epitope which binds to 8H9 could offer important
insight into the biology of these tumors. These studies are
currently underway. Further, during the course of the studies
reported here, we sought to evaluate in a general sense, the
function of sarcoma cells selected with 8H9. We observed that
Ewing's sarcoma cells positively selected using 8H9 retain their
clonogenic properties and are able to be maintained in cell
culture. This property has the potential aid in the generation and
study of tumor cell lines derived from patients with pediatric
sarcomas, which is currently difficult in these tumors due to
limitations of tumor size and surgical accessibility of primary
tumors. We are currently investigating whether Ewing's sarcoma
cells derived from apheresis or bone marrow samples in patients
with metastatic disease which are positively selected and grown in
culture could provide a ready source of tumor samples for further
biologic study.
RT-PCR is a powerfully sensitive tool for use in monitoring minimal
residual disease MRD.sup.30. It remains unclear, however, whether
evidence of small amounts of residual tumor by molecular analysis
is predictive for relapse in solid tumors and data in the
literature is conflicting. de Alava et al. evaluated MRD in Ewing's
sarcoma patients and showed a correlation between PCR positivity
and disease relapse. In this report however, some patients remained
PCR positive without disease relapse .sup.3. Using real-time PCR,
it is now possible to quantitate starting template and compare
starting template amount between samples obtained at different
timepoints. Real-time quantitative PCR has been used as a tool to
monitor MRD in leukemia patients .sup.31, 32 and may be useful in
evaluation of disease response .sup.33 and in predicting relapse in
patients by the detection of increasing levels of tumor specific
transcript.
This is the first report of the use of real-time quantitative PCR
used to detect and quantify Ewing's sarcoma transcript. It is
possible that quantitative PCR could allow for further
identification of patients with a high risk of relapse by detection
of increasing amounts of Ewing's transcripts over time. However,
because contamination of peripheral blood by solid tumors is likely
to be relatively low (in the range of 1:10.sup.5-1:10.sup.6 in this
series), the sensitivity of this analysis must be very high in
order to allow for the detection of very low levels of circulating
tumor in patients with solid tumors. The level of sensitivity of
our technique reached 1 Ewing's sarcoma cell in 10 .sup.6 normal
cells with nested PCR from 10.times.10.sup.6 cells. It is possible
that the level of sensitivity would be even higher if higher cell
numbers were evaluated since this method appears capable in our
hands of amplifying product from 10 contaminating cells. Tumor
enrichment using positive selection is another method to increase
sensitivity of tumor detection. The positive immunomagnetic
selection procedure described in this paper for purging could also
provide a suitable approach for tumor enrichment in for monitoring
MRD or even in contributing to making the correct diagnosis at the
time of initial presentation with metastatic disease. Indeed, cells
eluted from the column were positive by PCR analysis, demonstrating
the feasibility of this technique for tumor enrichment which would
be predicted to increase the sensitivity of PCR detection of
contaminating Ewing's sarcoma in patient samples. One caveat which
should be noted is that the quantitative technique, relies on the
assumption that the level of expression of t(11;22) is consistent
among cell lines and patient samples. This, may not be the case,
however, and may lead to under or over estimation of the absolute
level of tumor burden when comparing patient samples to a standard
curve. Such limitations would not preclude evaluation of changes in
the level of PCR positivity of an individual patient over time,
wherein substantial changes in the level of expression of t(11;22)
may be less likely.
In this report we have demonstrated a purging technique that
reduces tumor burden in artificially contaminated products by at
least 2-3 logs. This approach is predicted to substantially reduce
the tumor burden contained in autologous cellular products which
are administered in the context of innovative therapies for Ewing's
sarcoma. The demonstration that CFU assays on progenitor cells as
well as CD3 induced T cell proliferation are normal after purging
demonstrates no detrimental effects on normal progenitor cell and T
cell function, making this a potentially feasible addition to
autologous protocols. We conclude that immunomagnetic purging via
negative selection using MoAb 8H9 warrants evaluation in clinical
trials for Ewing's sarcoma involving the use of autologous
products.
REFERENCES
1. Arndt C A, Crist W M. Common musculoskeletal tumors of childhood
and adolescence. N Engl J Med. 1999; 341:342-52. 2. West D C, Grier
H E, Swallow M M, Demetri G D, Granowetter L, Sklar J. Detection of
circulating tumor cells in patients with Ewing's sarcoma and
peripheral primitive neuroectodermal tumor. J Clin Oncol. 1997;
15:583-8. 3. de Alava E, Lozano M D, Patino A, Sierrasesumaga L,
Pardo-Mindan F J. Ewing family tumors: potential prognostic value
of reverse-transcriptase polymerase chain reaction detection of
minimal residual disease in peripheral blood samples. Diagn Mol.
Pathol. 1998; 7:152-7. 4. Toretsky J A, Neckers L, Wexler L H.
Detection of (11;22)(q24;q12) translocation-bearing cells in
peripheral blood progenitor cells of patients with Ewing's sarcoma
family of tumors. J Natl Cancer Inst. 1995; 87:385-6. 5. Burdach S,
Jurgens H, Peters C, et al. Myeloablative radiochemotherapy and
hematopoietic stem-cell rescue in poor-prognosis Ewing's sarcoma. J
Clin Oncol. 1993; 11:1482-8. 6. Burdach S, Nurnberger W, Laws H J,
et al. Myeloablative therapy, stem cell rescue and gene transfer in
advanced Ewing tumors. Bone Marrow Transplant. 1996; 18 Suppl
1:S67-8. 7. Chan K W, Petropoulos D, Choroszy M, et al. High-dose
sequential chemotherapy and autologous stem cell reinfusion in
advanced pediatric solid tumors. Bone Marrow Transplant. 1997;
20:1039-43. 8. Fischmeister G, Zoubek A, Jugovic D, et al. Low
incidence of molecular evidence for tumour in PBPC harvests from
patients with high risk Ewing tumours. Bone Marrow Transplant.
1999; 24:405-9. 9. Frohlich B, Ahrens S, Burdach S, et al.
[High-dosage chemotherapy in primary metastasized and relapsed
Ewing's sarcoma. (EI)CESS]. Klin Padiatr. 1999; 211:284-90. 10.
Horowitz M E, Kinsella T J, Wexler L H, et al. Total-body
irradiation and autologous bone marrow transplant in the treatment
of high-risk Ewing's sarcoma and rhabdomyosarcoma. J Clin Oncol.
1993; 11:1911-8. 11. Ladenstein R, Lasset C, Pinkerton R, et al
Impact of megatherapy in children with high-risk Ewing's tumours in
complete remission: a report from the EBMT Solid Tumour Registry
[published erratum appears in Bone Marrow Transplant 1996
September; 18(3):675]. Bone Marrow Transplant. 1995; 15:697-705.
12. Ladenstein R, Philip T, Gardner H. Autologous stem cell
transplantation for solid tumors in children. Curr Opin Pediatr.
1997; 9:55-69. 13. Laws H J, Burdach S, van Kaick B, et al.
Multimodality diagnostics and megatherapy in poor prognosis Ewing's
tumor patients. A single-center report. Strahlenther Onkol. 1999;
175:488-94. 14. Pape H, Laws H J, Burdach S, et al. Radiotherapy
and high-dose chemotherapy in advanced Ewing's tumors. Strahlenther
Onkol. 1999; 175:484-7. 15. Perentesis J, Katsanis E, DeFor T,
Neglia J, Ramsay N. Autologous stem cell transplantation for
high-risk pediatric solid tumors. Bone Marrow Transplant. 1999;
24:609-15. 16. Pession A, Prete A, Locatelli F, et al. Phase I
study of high-dose thiotepa with busulfan, etoposide, and
autologous stem cell support in children with disseminated solid
tumors. Med Pediatr Oncol. 1999; 33:450-4. 17. Stewart D A, Gyonyor
E, Paterson A H, et al. High-dose melphalan+/-total body
irradiation and autologous hematopoietic stem cell rescue for adult
patients with Ewing's sarcoma or peripheral neuroectodermal tumor.
Bone Marrow Transplant. 1996; 18:315-8. 18. Rill D R, Santana V M,
Roberts W M, et al. Direct demonstration that autologous bone
marrow transplantation for solid tumors can return a multiplicity
of tumorigenic cells. Blood. 1994; 84:380-3. 19. Brenner M K, Rill
D R, Moen R C, et al. Gene-marking to trace origin of relapse after
autologous bone-marrow transplantation. Lancet. 1993; 341:85-6. 20.
Mackall C, Long L, Dagher R, et al. Combined Immune
Reconstitution/Tumor Vaccination to induce anti-tumor immune
responses in the setting of minimal residual neoplastic disease
[abstract]. Blood. 1999; 94:133a. 21. Quinones R R, Gutierrez R H,
Dinndorf P A, et al. Extended-cycle elutriation to adjust T-cell
content in HLA-disparate bone marrow transplantation. Blood. 1993;
82:307-17. 22. Kontny H U, Lehmbecher T M, Chanock S J, Mackall C
L. Simultaneous expression of Fas and nonfunctional Fas ligand in
Ewing's sarcoma. Cancer Res. 1998; 58:5842-9. 23. Chirgwin J M,
Przybyla A E, MacDonald R J, Rutter W J. Isolation of biologically
active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry. 1979; 18:5294-9. 24. de Wynter E A, Coutinho L H, Pei
X, et al. Comparison of purity and enrichment of CD34+ cells from
bone marrow, umbilical cord and peripheral blood (primed for
apheresis) using five separation systems. Stem Cells. 1995;
13:524-32. 25. Mackall C L, Gress R E. Pathways of T-cell
regeneration in mice and humans: implications for bone marrow
transplantation and immunotherapy. Immunol Rev. 1997; 157:61-72.
26. Dworzak M N, Fritsch G, Buchinger P, et al. Flow cytometric
assessment of human MIC2 expression in bone marrow, thymus, and
peripheral blood. Blood. 1994; 83:415-25. 27. Modak S, Gultekin S,
Kramer K, et al. Novel Tumor-Associated Antigen: Broad distribution
among neuroectodermal, mesenchymal and epithelial tumors, with
restricted distribution in normal tissues. Abstract: Proceedings at
ASCO. 1998. 28. Vogel W, Scheding S, Kanz L, Brugger W. Clinical
applications of CD34(+) peripheral blood progenitor cells (PBPC).
Stem Cells. 2000; 18:87-92. 29. Dyson P G, Horvath N, Joshua D, et
al. CD34+ selection of autologous peripheral blood stem cells for
transplantation following sequential cycles of high-dose therapy
and mobilisation in multiple myeloma [In Process Citation]. Bone
Marrow Transplant. 2000; 25:1175-84. 30. Emig M, Saussele S, Wittor
H, et al. Accurate and rapid analysis of residual disease in
patients with CML using specific fluorescent hybridization probes
for real time quantitative RT-PCR. Leukemia. 1999; 13:1825-32. 31.
Mensink E, van de Locht A, Schattenberg A, et al. Quantitation of
minimal residual disease in Philadelphia chromosome positive
chronic myeloid leukaemia patients using real-time quantitative
RT-PCR. Br J Haematol. 1998; 102:768-74. 32. Pongers-Willemse M J,
Verhagen O J, Tibbe G J, et al. Real-time quantitative PCR for the
detection of minimal residual disease in acute lymphoblastic
leukemia using junctional region specific TaqMan probes. Leukemia.
1998; 12:2006-14. 33. Branford S, Hughes T P, Rudzki Z. Monitoring
chronic myeloid leukaemia therapy by real-time quantitative PCR in
blood is a reliable alternative to bone marrow cytogenetics. Br J
Haematol. 1999; 107:587-99.
Fourth Series of Experiments
Disialoganglioside GD2 and Novel Tumor-Restricted Antigen 8H9:
Potential Targets for Antibody-Based Immunotherapy Against
Desmoplastic Small Round Cell Tumor.
Desmoplastic small round cell tumor (DSRCT) is an aggressive, often
misdiagnosed neoplasm of children and young adults. It is
chemotherapy-sensitive, yet patients often relapse off therapy
because of residual microscopic disease at distant sites:
peritoneum, liver, lymph node and lung. Strategies directed at
minimal residual disease (MRD) may be necessary for cure.
Monoclonal antibodies selective for cell surface tumor-associated
antigens may have utility for diagnosis and therapy of MRD, as
recently demonstrated in advanced-stage neuroblastoma (JCO 16:
3053, 1998). Using immunohistochemistry, we studied the expression
of two antigens: (1) G.sub.D2 using antibody 3F8 and (2) a novel
antigen using antibody 8H9, in a panel of 36 freshly frozen DSRCT.
G.sub.D2 is a disialoganglioside which is widely expressed among
neuroectodermal tumors as well as adult sarcomas. 8H9 recognizes a
surface 58 kD antigen expressed among neuroectodermal, mesenchymal
and epithelial tumors with restricted expression on normal tissues.
27 of 37 tumors (73%) were reactive with 3F8, and 35 of 37 (95%)
with 8H9. Both G.sub.D2 and the 58 kD antigen were found on tumor
cell membrane and in stroma. In general, immunoreactivity was
stronger and more homogeneous with 8H9 than with 3F8. These
antigens are potential targets for immunodiagnosis and
antibody-based therapy of DSRCT.
Desmoplastic small round cell tumor (DSRCT) is an aggressive,
ill-understood tumor affecting children and young adults. It is
characterized clinically by widespread abdominal serosal
involvement, metastasizes to peritoneum, liver, lungs and lymph
nodes, and is associated with a poor prognosis (Gerald et al.,
1991). Histologically, it consists of small, undifferentiated round
cells surrounded by an abundant desmoplastic stroma.
Immunohistochemically, the coexpression of epithelial, neural and
muscle markers is typical (Ordonez et al., 1993). DSRCT is
associated with a specific chromosomal translocation,
t(11;22)(p13;q12). The fused gene product aligns the NH2 terminal
domain of the EWS gene to the zinc finger DNA-binding domain of the
WT1 gene and is diagnostic of DSRCT (Ladanyi et al., 1994). This
fusion results in the induction of endogenous platelet derived
growth factor-A which stimulates fibroblast growth and may
contribute to the unique fibrosis observed with this tumor (Lee et
al, 1997). Further evidence of upregulation of growth factors
includes the reported expression of IGF-II, PDGF-.alpha. receptor
and IL-11 in DSRCT (Froberg et al., 1999).
Although dramatic response to aggressive multimodality therapy has
been demonstrated in the patients with DSRCT (Kushner et al.,
1996), many patients relapse with recurrent local disease or
distant metastases. Strategies aimed at eradication of MRD are,
therefore, warranted in the management of patients with DSRCT.
Monoclonal antibodies selective for cell surface tumor-associated
antigens are potential candidates as recently demonstrated in
neuroblastoma where immune targeting of the diasialoganglioside
G.sub.D2 has significantly improved long-term survival in patients
with stage 4 disease (Cheung et al., 1998). Few such
tumor-associated targets have been defined for DSRCT. We describe
here two possible targets for such immunotherapy: G.sub.D2 targeted
by the monoclonal antibody 3F8 and a novel tumor antigen recognized
by the monoclonal antibody 8H9.
Materials and Methods
Tumor and Normal Tissue Samples
Frozen tumors from 37 patients with DSRCT were analyzed. Diagnosis
was confirmed by hematoxylin and eosin assessment of paraffin-fixed
specimens.
Monoclonal Antibodies
The murine IgG.sub.3 monoclonal antibody 3F8 was purified from
ascites as previously described (Cheung et al., 1985). Using a
similar technique, female BALB/c mice were hyperimmunized with
human neuroblastoma. Lymphocytes derived from these mice were fused
with SP2/0 mouse myeloma cells line. Clones were selected for
specific binding on ELISA. The 8H9 hybridoma secreting an IgG.sub.1
monoclonal antibody was selected. 8H9 was produced in vitro and
purified by protein G (Pharmacia, Piscataway, N.J.) affinity
chromatography.
Immunohistochemical Studies
Eight .mu.m cryostat frozen tumor sections were fixed in acetone
and washed in PBS. Immunohistochemical studies were performed as
previously described (Kramer et al. 1996) Endogenous peroxidases
were blocked in 0.3% H.sub.2O.sub.2 in PBS. Sections were incubated
in 10% horse serum (Gibco BRL Gaithersburg, Md.) after blocking
with avidin and biotin. Incubation with purified 8H9 diluted in PBS
to 2 .mu.g/ml was carried out at room temperature for 1 hour. An
IgG1 myeloma was used as a control (Sigma Chemical, St Louis Mo.).
Sections were incubated with a secondary horse anti-mouse
biotinylated antibody (Vector Laboratories, Burlingame, Calif.)
followed by incubation with ABC complex (Vector Laboratories,
Burlingame, Calif.) and stained with Vector VIP peroxidase
substrate (Vector Laboratories, Burlingame, Calif.) or DAB
peroxidase substrate kit (Vector Laboratories, Burlingame, Calif.).
A 10% hematoxylin counterstain for 2 minutes was used. Staining was
graded as positive or negative and homogenous or heterogenous
reactivity noted.
Results
Clinical Profile
Of the 37 patients studied, 32 were male and five female. Age at
diagnosis ranged from 13 to 46 years (median 18 years). All
received treatment with an aggressive multimodality regimen
including dose-intensive chemotherapy.
Immunoreactivity
Tumor sections from 37 patients were tested for the expression of
G.sub.D2 and the antigen recognized by 8H9 by immunohistochemistry.
27 of 37 (73%) tested positive for G.sub.D2. (Table 1). Most tumors
had strong immunoreactivity (>1+) Immunoreactivity was seen
homogeneously in most tumors and was localized to the cell membrane
(FIG. 16). Intense stromal staining was marked in all tumors
studied.
TABLE-US-00020 TABLE 1 Immunoreactivity of 3F8 and 8H9 with DSRCT
No. Reactivity No. Marker tested 0 1+ 2+ 3+ pos. (%) Homogeneous
Heterogeneous G.sub.D2 36 10 10 12 4 26 (72) 19 7 Antigen 8H9 36 2
9 17 8 34 (94) 32 2
35 of 37 (95%) tumors tested positive for 8H9 Immunoreactivity had
a characteristic cell membrane localization and was homogeneous in
almost all tumors (FIG. 17). Immunoreactivity was more strongly
marked than that with 3F8. Equally strong stromal staining was
seen.
Clinicopathologic Correlation
In this group of highly aggressive disseminated tumors, there was
no correlation between outcome and the expression of either
G.sub.D2 or the 8H9 antigen (Table 2)
TABLE-US-00021 TABLE 2 G.sub.D2 and Antigen 8H9: Correlation with
outcome G.sub.D2 positive 8H9 positive Expired* 10/17 16/17
Survivors < 18 mo since diagnosis 11/14 13/14 Survivors > 18
mo since diagnosis 5/5 5/5 *1 Patient died of treatment-related
toxicity
Discussion
The clinicopathological spectrum of DSRCT continues to be further
defined since the initial series was reported in 1991 (Gerald et
al., 1991). Chemosensitivity to doxorubicin and alkylator-based
chemotherapy has been reported (Gonzalez-Crussi et al., 1990).
Prolonged survival in response to an aggressive multimodality
regimen including high-dose chemotherapy, radiation and surgery has
also been reported (Kushner et al., 1996). However, most patients
succumb to recurrent local disease or metastases to peritoneum,
liver, lymph nodes, or lung. Relapses can be largely attributed to
the failure of eradication of MRD. Alternative therapeutic
strategies to target MRD are therefore warranted. One such strategy
could be directed at the upregulated growth factors particularly
PDGFA and related factors expressed on DSRCT (Froberg et al.,
1999). Targeted immunotherapy utilizing monoclonal antibodies,
which does not add to the toxicity of chemotherapy, is another
approach.
DSRCT is characterized by the coexpression of epithelial,
mesenchymal and neuroectodermal markers. Recent publications have
defined the immunohistochemical and molecular make-up of DSRCT
(Ordonez, 1998; Gerald, 1999). However, most of the markers
identified cannot be used as targets for antibody mediated
immunotherapy either due to crossreactivity with normal tissues or
inaccessibility to monoclonal antibodies due to localization in the
nucleus or cytoplasm. (Table 3). The most commonly expressed
markers on DSRCT including desmin, cytokeratin, vimentin,
epithelial membrane antigen and neuron-specific enolase are also
widely expressed on normal tissues. The MIC2 antigen has been
reported to be expressed on 20-35% of DSRCT. However, unlike
Ewing's sarcoma family of tumors, which have membrane localization,
immunoreactivity in DSRCT is primarily cytoplasmic (Gerald et al,
1998). MOC31, a monoclonal antibody that recognizes epithelial
glycoprotein 2 (EGP-2) has been shown to be reactive with most
DSRCT tested (Ordonez, 1998). EGP-2 is overexpressed on epithelial
tumors, but is also present on normal epithelial cells (de Leij et
al, 1994). Antibodies directed against the WT1 protein have strong,
specific, nuclear immunoreactivity with almost all DSRCT tested
(Gerald et al, 1998)
TABLE-US-00022 TABLE 3 Previously reported antigens on DSRCT
Antigen Localization Crossreactivity Intermediate filaments Desmin
cytoplasm skeletal, cardiac & smooth muscle Vimentin cytoplasm
mesenchymal tissues Keratin cytoplasm epithelial cells Epithelial
antigens Epithelial membrane antigen cytoplasm epithelial cells
Epithelial glycoprotein-2 cytoplasm epithelial cells Ber-Ep4
antigen cytoplasm epithelial cells Neural antigens CD57 cytoplasm
neural tissues Neuron-specific enolase cytoplasm neural tissues
MIC-2 cytoplasm & cell membrane lymph nodes, epithelial cells
WT1 protein Nucleus None PDGFA Cell membrane Endothelial cells,
PDGF-.alpha.receptor Cell membrane hematopoeitic cells Endothelial
cells, hematopoeitic cells
The reported expression of neuroectodermal antigens on DSRCT led us
to study these tumors for the expression of G.sub.D2: a
disialoganglioside which is expressed on other small blue round
cell tumors such as neuroblastoma, small cell lung cancer, melanoma
and osteosarcoma (Heiner et al., 1987) as well as on adult soft
tissue sarcomas (Chang et al., 1992). G.sub.D2 is a safe target for
immunotherapy based on clinical trials of the anti-G.sub.D2
antibody 3F8 in patients with neuroblastoma. tissues of the nervous
system (Cheung et al., 1998). Serum G.sub.D2 does not interfere
with the biodistribution of specific antibodies and the antigen is
not modulated from the cell surface upon binding by antibodies.
Successful targeting of the monoclonal antibody 3F8 to G.sub.D2 was
previously demonstrated in neuroblastoma (Yeh et al., 1991) and
small cell lung cancer (Grant et al., 1996). 3F8 has also shown
efficacy in clinical trials in patients with neuroblastoma (Cheung
et al., 1998b) and melanoma (Cheung et al., 1987). Furthermore, 3F8
appeared to induce long-term remissions in patients with Stage 4
neuroblastoma. Reported side effects are short-lived and manageable
(Cheung et al., 1998). In our study 72% of DSRCT tested were
immunoreactive with the anti-GD2 antibody 3F8. Most tumors showed
strong, homogeneous reactivity localized to the cell membrane.
(Table 1) (FIG. 16) DSRCT may be a putative tumor for in vivo
antibody targeting with 3F8. Alternatively, an anti-idiotypic
vaccine approach can be utilized as has been suggested for
neuroblastoma. (Cheung et al, 1994)
The monoclonal antibody 8H9 is a murine IgG1 derived from mice
immunized with neuroblastoma. It has been shown to have a broad
expression on neuroectodermal, mesenchymal and epithelial tumors
with limited expression on normal tissues. (data not shown). Its
immunoreactive profile led us to use it for testing DSRCT. 95% of
tumors tested positive with DSRCT Immunoreactivity with DSRCT was
localized to the stroma and cell membrane (FIG. 17) and for most
tumors was intense and homogeneous, and in general, stronger than
that observed for GD2 (Table 2).
The target antigen for 8H9 appears to be a novel 58 kD glycoprotein
with a unique distribution on cell membranes of tumors of varying
lineage, but restricted expression in normal tissues. This tissue
distribution makes it likely to be a unique antigen not previously
described on DSRCT. The cell membrane localization of 8H9 allows it
to be targeted by monoclonal antibodies. 8H9 conjugated with
1.sup.131 has been shown to radioimmunolocalize neuroblastoma and
rhabdomyosarcoma xenografts in mice without significant
crossreactivity with other organs. (data not shown).
In the therapy of DSRCT, strategies to eliminate minimal residual
disease are necessary to produce cures. Monoclonal antibody based
therapy may augment aggressive multimodality therapy by targeting
minimal residual disease without adding to toxicity. Our study has
identified G.sub.D2 and antigen 8H9 as two hitherto undescribed
markers for DSRCT, which can potentially be targets for
differential diagnosis and immunotherapy.
REFERENCES
Chang H. R., Cordon-Cardo C., Houghton A. N., Cheung N. K., and
Brennan M. F., Expression of disialogangliosides G.sub.D2 and
G.sub.D3 on human soft tissue sarcomas. Cancer 70: 633-8, (1992)
Cheung N. K., Saarinen, U., Neely, J., Landmeier, B., Donovan D.,
and Coccia, P. Monoclonal antibodies to a glycolipid antigen on
human neuroblastoma cells. Cancer Res., 45: 2642-2649, (1985)
Cheung N. K., Lazarus, H., Miraldi F. D., Abramowsky, C. R.,
Kallick S., Saarinen, U. M., Spitzer, T., Strandjord, S. E.,
Coccia, P. F., and Berger, N. A. Ganglioside G.sub.D2 specific
monoclonal antibody 3F8: a phase I study in patients with
neuroblastoma and malignant melanoma. J. Clin. Oncol. 5: 1430-40,
(1987) Cheung, N. K., Cheung, I. Y., Canete, A., Yeh, S. J.,
Kushner, B., Bonilla, M. A., Heller, G., and Larson, S. M. Antibody
response to murine anti G.sub.D2 monoclonal antibodies: correlation
with patient survival. Cancer Res. 54: 2228-33 (1994) Cheung N K.,
Kushner B. H., Yeh S. D. J., and Larson S. M., 3F8 monoclonal
antibody treatment of patients with stage 4 neuroblastoma: a phase
II study. Int. J. Oncol 12: 1299-306, (1998b) Cheung, N. K.,
Kushner, B. H., Cheung, I. Y., Kramer, K., Canete, A., Gerald, W.,
Bonilla, M. A., Finn, R., Yeh, S., and Larson, S. M., Anti G.sub.D2
antibody treatment of minimal residual stage 4 neuroblastoma
diagnosed at more than 1 year of age. J. Clin. Oncol., 16: 3053-60,
(1998) De Leij, L., Helrich, W., Stein, R., and Mattes M. J.
SCLC-cluster-2 antibodies detect the pancarcinoma/epithelial
glycoprotein E GP-2 (supplement) Int. J. Cancer 8: 60-3, 1994
Froberg, K., Brown, R. E., Gaylord, H., Manivel, C.,
Intra-abdominal desmoplastic small round cell tumor:
immunohistochemical evidence for up-regulation of autocrine and
paracrine growth factors. Ann Clin Lab Sci 29: 78-85, 1999
Gonzalez-Crussi, F., Crawford, S. E., and Sun, C. J. Intraabdominal
desmoplastic small-cell tumors with divergent differentiation.
Observation on three cases of childhood. Am. J. Surg. Pathol. 15:
499-513, (1991) Grant, S. C., Kostakoglu, L., Kris, M. G., Yeh, S.
D., Larson, S. M., Finn, R. D., Oettgen, H. F., and Cheung, N. K.
targeting of small-cell lung cancer using the anti-G.sub.D2
ganglioside monoclonal antibody 3F8: a pilot trial. Eur. J. Nucl.
Med. 23: 145-9 (1996) Heiner, J. P., Miraldi, F., Kallick, S.,
Makley J., Neely, J., Smith-Mensah, W. H., and Cheung N. K.
Localization of G.sub.D2-specific monoclonal antibody 3F8 in human
osteosarcoma. Cancer Res. 47: 5377-81 (1987) Kramer, K., Gerald,
W., Le Sauteur, L., UriSaragovi, H., and Cheung, N. K. Prognostic
Value of TrkA Protein Detection by Monoclonal Antibody 5C3 in
Neuroblastoma. Clin. Cancer Res. 2: 1361-1367, 1996 Kushner, B. H.,
LaQuaglia M. P., Wollner, N., Meyers, P. A., Lindsley, K. L.,
Ghavimi, F., Merchant, T. E., Boulad, F., Cheung, N. K., Bonilla,
M. A., Crouch, G., Kelleher, J. F., Steinherz, P. G., and Gerald,
W. L., Desmoplastic small round-cell tumor: prolonged
progression-free survival with aggressive multimodality therapy. J.
Clin. Oncol. 14: 1526-31, (1996) Ladanyi, M., and Gerald, W.,
Fusion of the EWS and WT1 genes in the desmoplastic small round
cell tumor. Cancer Res. 54: 2837-40, (1994) Lee, S. B., Kolquist,
K. A., Nichols, K., Englert, C., Maheshwaran, S., Ladanyi, M., et
al., The EWS-WT1 translocation product induces PDGFA in
desmoplastic small round-cell tumour. Nat Genet. 17, 309-13, 1997
Gerald, W. L., Miller, H. K., Battifora, H., Miettenen, M., Silva,
E. G., and Rosai, J., Intrabdominal desmoplastic small round cell
tumor. Report of 19 cases of a distinctive type of high-grade
polyphenotypic malignancy affecting young individuals. Am. L. Surg.
Pathol. 15, 499-513, (1991) Gerald, W. L., Ladanyi, M. L., De
Alava, E., Cuatrecasas, M., Kushner, B. H., LaQuaglia, M. P., and
Rosai, J. Clinical pathologic, and molecular spectrum of tumors
associated with t(11;22)(p13;q12): desmoplastic small round-cell
tumor and its variants. J. Clin. Oncol., 16: 3028-36, (1998)
Ordonez, N. G., El-Naggar, A. K., Ro, J. Y., Silva, E. G., Mackay
B., Intra-abdominal desmoplastic small cell tumor: a light
microscopic, immunocytochemical, ultrastructural, and flow
cytometric study. Hum. Pathol. 24, 850-65, (1993) Ordonez, N. G.
Desmoplastic small round cell tumor: II: an ultrastructural and
immunohistochemical study with emphasis on new immunohistochemical
markers. Am. J. Surg. Pathol. 22: 1314-27, (1998) Yeh S. D.,
Larson, S. M., Burch, L., Kushner, B. H., LaQuaglia, M, Finn, R.,
and Cheung, N. K. Radioimmunodetection of neuroblastoma with
iodine-131-3F8: correlation with biopsy,
iodine-131-metaiodobenzylguanidine and standard diagnostic
modalities. J. Nucl. Med. 32: 769-76 (1991)
Fifth Series of Experiments
Anti-Idiotypic Antibody as the Surrogate Antigen for Cloning ScFv
and its Fusion Proteins
ScFv provides a versatile homing unit for novel antibody-fusion
constructs. However, a reliable screening and binding assay is
often the limiting step for antigens that are difficult to clone or
purify. We demonstrate that anti-idiotypic antibodies can be used
as surrogate antigens for cloning scFv and their fusion proteins.
8H9 is a murine IgG1 monoclonal antibody specific for a novel
antigen expressed on the cell surface of a wide spectrum of human
solid tumors but not in normal tissues (Cancer Res 61:4048, 2001)
Rat anti-8H9-idiotypic hybridomas (clones 2E9, 1E12 and 1F11) were
produced by somatic cell fusion between rat lymphocytes and mouse
SP2/0 myeloma. In direct binding assays (ELISA) they were specific
for the 8H9 idiotope. Using 2E9 as the surrogate antigen, 8H9-scFv
was cloned from hybridoma cDNA by phage display. 8H9scFv was then
fused to .quadrature.human-1-CH2-CH3 cDNA for transduction into CHO
and NSO cells. High expressors of mouse scFv-human Fc chimeric
antibody were selected. The secreted homodimer reacted specifically
with antigen-positive tumor cells by ELISA and by flow cytometry,
inhibitable by the anti-idiotypic antibody. The reduced size
resulted in a shorter half-life in vivo, while achieving comparable
tumor to nontumor ratio as the native antibody 8H9. However, it
could not mediate antibody-dependent cell-mediated or
complement-mediated cytotoxicities in vitro.
1. Introduction
The ability to condense the binding site by genetic fusion of
variable region immunoglobulin genes to form scFv has greatly
expanded the potential and development of antibody-based targeted
therapies (Bird et al., 1988; Huston et al., 1988; Winter and
Milstein, 1991; George et al., 1994). Using phage display
libraries, scFv can now be cloned from cDNA libraries derived from
rodents, immunized volunteers, or patients (Burton and Barbas III,
1994; Winter et al., 1994; Cai and Garen, 1995; Raag and Whitlow,
1995). The availability of hIg-transgenic and transchromosomal mice
will allow immunization schema or pathogens not feasible or safe in
humans. Construction of the scFv is the critical first step in the
synthesis of various fusion proteins, including scFv-cytokine (Shu
et al., 1993), scFv-streptavidin (Kipriyanov et al., 1995),
scFv-enzyme (Michael et al., 1996), scFv-toxins (Wikstrand et al.,
1995), bispecific scFv (diabodies) (Alt et al., 1999), bispecific
chelating scFv (DeNardo et al., 1999), scFv-Ig (Shu et al., 1993),
tetravalent scFv (Alt et al., 1999; Santos et al., 1999) and
scFv-retargeted T-cells (Eshhar et al., 1993). ScFv-Ig constructs
mimic natural IgG molecules in their homodimerization through the
Fc region, as well as their ability to activate complement (CMC)
and mediate antibody dependent cell-mediated cytotoxicites
(ADCC).
The construction of scFv requires a reliable antigen preparation
both for panning phages and for binding assays. They often become a
rate-limiting step (Lu and Sloan, 1999), particularly for antigens
that are difficult to clone or purify. Cell-based phage display
(Watters et al., 1997), and enzyme linked immunosorbent assays
(ELISA) when optimized, have been successfully applied as
alternatives. However, subtle differences in the panning step can
determine the success or failure of phage display (Tur et al.,
2001). For example, a reduction in wash pH is needed for scFv
directed at ganglioside GD2 in order to reduce nonspecific
adherence of phage particles (Tur et al., 2001). Moreover, phage
binding assay may require membrane preparations to withstand the
vigorous washing procedure.
Anti-idiotypic antibodies are frequently used as antigen mimics of
infectious agents and tumor antigens (Thanavala et al., 1986;
Wagner et al., 1997). When made as MoAb, they are ideal surrogates
when the target antigen is not readily available. The
physico-chemical behavior of immunoglobulins as antigens in panning
and binding assays is generally known and can be easily
standardized. We recently described a novel tumor antigen reactive
with a murine MoAb 8H9 (Modak et al., 2001). Given its lability and
glycosylation, this antigen is difficult to purify. Here we
describe the use of an anti-idiotypic antibody as a surrogate
antigen for cloning a scFv derived from the 8H9 hybridoma cDNA
library, and for the selection of chimeric mouse scFv-human Fc
fusion constructs.
2. Materials and Methods
2.1 Animals
BALB/c mice were purchased from Jackson Laboratories, Bar Harbor,
Me. Lou/CN rats were obtained from the National Cancer
Institute-Frederick Cancer Center (Bethesda, Md.) and maintained in
ventilated cages. Experiments were carried out under a protocol
approved by the Institutional Animal Care and Use Committee, and
guidelines for the proper and humane use of animals in research
were followed.
2.2 Cell Lines
Human neuroblastoma cell lines LAN-1 was provided by Dr. Robert
Seeger (Children's Hospital of Los Angeles, Los Angeles, Calif.),
and NMB7 by Dr. Shuen-Kuei Liao (McMaster University, Ontario,
Canada). Cell lines were cultured in 10% defined calf serum
(Hyclone, Logan, Utah) in RPMI with 2 mM L-glutamine, 100 U/ml of
penicillin (Sigma-Aldrich, St. Louis, Mo.), 100 ug/ml of
streptomycin (Sigma-Aldrich), 5% CO.sub.2 in a 37.degree. C.
humidified incubator. Normal human mononuclear cells were prepared
from heparinized bone marrow samples by centrifugation across a
Ficoll-Hypaque density separation gradient. Human AB serum (Gemini
Bioproducts, Woodland, Calif.) was used as the source of human
complement.
2.3 Monoclonal Antibodies
Cells were cultured in RPMI 1640 with 10% newborn calf serum
(Hyclone, Logan, Utah) supplemented with 2 mM glutamine, 100 U/ml
of penicillin and 100 ug/ml of streptomycin (Sigma-Aldrich). 3F8,
an IgG3 MoAb raised in a Balb/c mouse against human neuroblastoma,
specifically recognizes the ganglioside GD2. The BALB/c myeloma
proteins MOPC-104E, TEPC-183, MOPC-351, TEPC-15, MOPC-21, UPC-10,
MOPC-141, FLOPC-21, and Y5606 were purchased from Sigma-Aldrich.
MoAb R24 (anti-GD3), V1-R24, and K9 (anti-GD3) were gifts from Dr.
A. Houghton, OKB7 and M195 (anti-CD33) from Dr. D. Scheinberg, and
10-11 (anti-GM2) from Dr. P. Livingston of Memorial Sloan Kettering
Cancer Center, New York; and 528 (EGF-R) from Dr. J. Mendelsohn of
MD Anderson, Houston, Tex. 2E6 (rat anti-mouse IgG3) was obtained
from hybridomas purchased from American Type Culture Collection
[ATCC] (Rockville, Md.). NR-Co-04 was provided by Genetics
Institute (Cambridge, Mass.). In our laboratory, 5F9, 8H9, 3A5,
3E7, 1D7, 1A7 were produced against human neuroblastoma; 2C9, 2E10
and 3E6 against human breast carcinoma, and 4B6 against
glioblastoma multiforme. They were all purified by protein A or
protein G (Pharmacia, Piscataway, N.J.) affinity
chromatography.
2.4 Anti-8H9 Anti-Idiotypic Antibodies
LOU/CN rats were immunized intraperitoneally (ip) with 8H9 (400 ug
per rat) complexed with rabbit anti-rat serum (in 0.15 ml), and
emulsified with an equal volume (0.15 ml) of Complete Freund's
Adjuvant (CFA) (Gibco-BRL, Gaithersburg, Md.). The 8H9-rabbit-IgG
complex was prepared by mixing 2 ml (8 mg) of purified 8H9 with 4
ml of a high titer rabbit anti-rat precipitating serum (Jackson
Immunoresearch Laboratories, West Grove, Pa.). After incubation at
4.degree. C. for 3 hours, the precipitate was isolated by
centrifugation at 2500 rpm for 10 minutes, and resuspended in PBS.
Three months after primary immunization, the rats were boosted ip
with the same antigen in CFA. One month later, a 400 ug boost of
8H9-rabbit-anti-mouse complex was injected intravenously. Three
days afterwards, the rat spleen was removed aseptically, and
purified lymphocytes were hybridized with SP2/0-Ag14 (ATCC). Clones
selection was based on specific binding to 8H9 and not to control
antibody 5F9, a murine IgG1. Repeated subcloning using limiting
dilution was done. Isotypes of the rat monoclonal antibodies were
determined by Monoclonal Typing Kit (Sigma-Aldrich). Rat
anti-idiotypic antibody clones (2E9, 1E12, 1F11) were chosen and
produced by high density miniPERM bioreactor (Unisyn technologies,
Hopkinton, Mass.), and purified by protein G affinity
chromatography (Hitrap G, Pharmacia). The IgG fraction was eluted
with pH 2.7 glycine-HCl buffer and neutralized with 1 M Tris buffer
pH 9. After dialysis in PBS at 4.degree. C. for 18 hours, the
purified antibody was filtered through a 0.2 um millipore filter
(Millipore, Bedford, Mass.), and stored frozen at -70.degree. C.
Purity was determined by SDS-PAGE electrophoresis using 7.5%
acrylamide gel.
The "standard" ELISA to detect rat anti-idiotypic antibodies (Ab2)
was as follows: Purified 8H9, or irrelevant IgG1 myeloma, were
diluted to 5 ug/ml in PBS and 50 ul per well was added to 96-well
flat-bottomed polyvinylchloride (PVC) microtiter plates and
incubated for 1 hour at 37.degree. C. Rows with no antigen were
used for background subtraction. Filler protein was 0.5% BSA in PBS
and was added at 100 ul per well, and incubated for 30 minutes at
4.degree. C. After washing, 50 ul duplicates of hybridoma
supernatant was added to the antigen-coated wells and incubated for
3 hours at 37.degree. C. The plates were washed and a
peroxidase-conjugated mouse anti-rat IgG+IgM (Jackson
Immunoresearch Laboratory) at 100 ul per well was allowed to react
for 1 hour at 4.degree. C. The plate was developed using the
substrate o-phenylenediamine (Sigma-Aldrich) (0.5 mg/ml) and
hydrogen peroxide (0.03%) in 0.1 M citrate phosphate buffer at pH
5. After 30 minutes in the dark, the reaction was quenched with 30
ul of 5 N sulfuric acid and read using an ELISA plate reader.
2.5 Specificity by Direct Binding Assay
Fifty ul per well of purified mouse monoclonal antibodies or
myelomas were coated onto 96-well PVC microtiter plates at 5 ug/ml
for 60 minutes at 37.degree. C., aspirated and then blocked with
100 ul of 0.5% BSA filler protein per well. After washing and
air-drying, the wells were allowed to react with anti-idiotypic
antibodies. The rest of the procedure was identical to that
described in the "standard" assay.
2.6 Specificity by Inhibition Assay
To further examine the specificity of these anti-idiotypic
antibodies, inhibition of 8H9 immunofluorescent staining of tumor
cells by anti-idiotypic antibodies was tested. Purified 8H9 and
anti-GD2 MoAb 3F8, (all 10 ug/ml in 0.5% BSA) were preincubated
with various concentrations of anti-idiotypic antibodies for 30
minutes on ice before reacting with 10.sup.6 cells of either
GD2-positive/8H9 positive LAN-1 (neuroblastoma) or
GD2-negative/8H9-positive HTB-82 (rhabdomyosarcoma). The cells were
then washed twice in PBS with 0.1% sodium azide and reacted with
FITC-conjugated rat anti-mouse IgG (Biosource, Burlingame, Calif.)
on ice for 30 minutes in the dark. The cells were washed in PBS
with azide, fixed in 1% paraformaldehyde and analyzed by FACScan
(Becton-Dickinson, CA). The mean fluorescence was calculated and
the inhibition curve computed.
2.7 Construction of scFv Gene
mRNA was isolated from 8H9 hybridoma cells using a commercially
available kit (Quick Prep Micro mRNA Purification, Pharmacia
Biotech) following the procedures outlined by the manufacturer.
5.times.10.sup.6 hybridoma cells cultured in RPMI-1640 medium
supplemented 10% calf serum, L-glutamine (2 mmol/L), penicillin
(100 u/L) and streptomycin sulphate (100 ug/ml) were pelleted by
centrifugation at 800.times.g and washed once in RNase-free
phosphate buffered saline (pH 7.4). The recentrifuged cells were
lysed directly in the extraction buffer. Poly(A)-RNA was purified
by a single fractionation over oligo (dT)-cellulose and eluted from
oligo (dT) cellulose in the elution buffer. The mRNA sample was
precipitated for 1 hour with 100 ug glycogen, 40 ul of 2M potassium
acetate solution and 1 ml of absolute ethanol at -20.degree. C. The
nucleic acid was recovered by centrifugation at 10,000.times.g for
30 min The sample was evaporated until dry, and dissolved in 20 ul
RNase-free water.
ScFv gene was constructed by recombinant phage display. 5 ul of
mRNA was reversely transcribed in a total volume of 11 ul reaction
mixture and 1 ul dithiothreitol (DTT) solution for 1 hour at
37.degree. C. For the PCR amplification of 8H9 immunoglobulin
variable regions, light chain primer mix and the heavy chain primer
set (Pharmacia) were added respectively to generate suitable
quantities of the heavy (340 bp) and light (325 bp) chain.
Following an initial 10 min dwell at 95.degree. C., 5 U AmpliTaq
Gold DNA polymerase (Applied Biosystems, Foster City, Calif.) was
added. The PCR cycle consisted of a 1 min denaturation step at
94.degree. C., a 2 min annealing step at 55.degree. C. and a 2 min
extension step at 72.degree. C. After 30 cycles of amplification,
PCR derived fragment was purified by the glassmilk beads (Bio101,
Vista, Calif.) and then separated by 1.5% agarose gel
electrophoresis in TAE buffer and detected by ethidium bromide
staining.
For the assembly and fill-in reaction, both purified heavy chain
and light chain fragments were added to an appropriate PCR mixture
containing a 15 amino acid linker-primer for 8H9, dNTPs, PCR buffer
and Ampli Taq Gold DNA polymerase. PCR reactions were performed at
94.degree. C. for 1 min, followed by a 4 min annealing reaction at
63.degree. C. The heavy and light chain DNA of 8H9 were joined by
the linker (GGGS).sub.3 (Pharmacia) into scFv in a VH-VL
orientation after 7 thermocycles.
Using an assembled scFv DNA of 8H9 as template, a secondary PCR
amplification (30 standard PCR cycles) was carried out using
primers containing either Sfi I or Not I restriction sites. Thus,
the Sfi I and Not I restriction sites were introduced to the 5' end
of heavy chain and the 3' end of light chain, respectively.
Amplified ScFv DNAs were purified by glassmilk beads and digested
with Sfi I and Not I restriction endonucleases. The purified ScFv
of 8H9 was inserted into the pHEN1 vector (kindly provided by Dr.
G. Winter, Medical Research Council Centre, Cambridge, UK)
containing Sfi I/Nco I and Not I restriction sites. Competent E.
coli XL 1-Blue cells (Stratagene, La Jolla, Calif.) were
transformed with the pHEN1 phagemid. Helper phage M13 KO7
(Pharmacia) was added to rescue the recombinant phagemid.
2.8 Enrichment of Recombinant Phagemid by Panning
50 ul of anti-8H9 idiotypic antibody 2E9 (50 ug/ml) in PBS was
coated on the 96-well PVC microtiter plates and incubated at
37.degree. C. for 1 hour. 100 ul of the supernatant from phage
library was added to each well and incubated for 2 hours. The plate
was washed 10 times with PBS containing 0.05% BSA. Antigen-positive
recombinant phage captured by the anti-idiotype MoAb 2E9 was eluted
with 0.1M glycine-HCl (pH 2.2 containing 0.1% BSA) and neutralized
with 2M Tris solution. This panning procedure was repeated three
times. The phagemid 8HpHM9F7-1 was chosen for the rest of the
experiments.
2.9 ELISA
The selected phage was used to reinfect E. coli XL 1-Blue cells.
Colonies were grown in 2.times.YT medium containing ampicillin (100
ug/ml) and 1% glucose at 30.degree. C. until the optical density of
0.5 unit at 600 nm was obtained. Expression of scFv antibody was
induced by changing to the medium containing 100 uM IPTG
(Sigma-Aldrich) and incubating at 30.degree. C. overnight. The
supernatant obtained from the medium by centrifugation was directly
added to the plate coated with anti-idiotype 2E9. The pellet was
resuspended in the PBS containing 1 mM EDTA and incubated on ice
for 10 min. The periplasmic soluble antibody was collected by
centrifugation again and added to the plate. After a 2-hour
incubation at 37.degree. C., plates were washed and anti-MycTag
antibody (clone 9E10 from ATCC) was added for 1 hour at 37.degree.
C. After washing, affinity purified goat anti-mouse antibody
(Jackson Immunoresearch) was allowed to react for 1 hour at
37.degree. C. and the plates were developed with the substrate
o-phenylenediamine (Sigma-Aldrich) as previously described.
2.10 Construction of ScFv-Human-.quadrature.1-CH2-CH3 Mouse
Human-Chimeric Gene
A single gene encoding scFv8H9 was generated by PCR method using
phagemid 8HpHM9F7-1 as the template. Secondary PCR amplification
(30 PCR cycles) was carried out to insert the human IgG1 leader
sequence at the 5' end of the scFv8H9 DNA plus the restriction
sites at the two opposite ends, i.e. Hind III and Not I, at the 5'
end of human IgG1 leader and at the 3' end of scFv8H9,
respectively. Amplified human IgG1 leader-scFv8H9 DNA was purified
by glassmilk beads and digested with Hind III and Not I restriction
endonucleases according to manufacturer's instructions. The Hind
III-Not I fragment of human IgG1 leader-scFv8H9 cDNA was purified
on agarose gel and ligated into pLNCS23 vector carrying the
human-(1-CH2-CH3 gene (kindly provided by Dr. J. Schlom, National
Cancer Institute, NIH, Bethesda, Md.) (Shu et al., 1993). Competent
E. coli XL 1-Blue cells were transformed with pLNCS23 containing
the scFv phagemid. The scFv-CH2-CH3 DNA was primed with appropriate
primers and sequenced using the Automated Nucleotide Sequencing
System Model 373 (Applied Biosystems). The sequences agreed with
the cDNA sequences of the light and heavy chains of 8H9 as well as
the human-.quadrature.1-CH2-CH3 available from GenBank, including
the ASN 297 of the CH2 domain. In this construct, Cys220 of the
genetic hinge was replaced by a proline residue, while Cys226 and
Cys229 were retained in the functional hinge (Shu et al., 1993)
2.11 Cell Culture and Transfection
CHO cell or NSO myelomas cells (Lonza Biologics PLC, Bershire, UK)
were cultured in RPMI 1640 (Gibco-BRL) supplemented with glutamine,
penicillin, streptomycin (Gibco-BRL) and 10% fetal bovine serum
(Gibco-BRL). Using effectene transfection reagent (Qiagen,
Valencia, Calif.), recombinant ScFv8H9-human-.quadrature.1-CH2-CH3
was introduced via the pLNCS23 into CHO cell or NSO myelomas cells.
Cells were fed every 3 days, and G418 (1 mg/ml; Gibco-BRL)
resistant clones were selected. After subcloning by limiting
dilution, chimeric antibodies were produced by high density
miniPERM bioreactor from Unisyn Technologies using 0.5% ULG-FBS in
Hydridoma-SFM (Invitrogen Corporation, Carlsbad, Calif.). The
chimeric antibodies were purified by protein G (Pharmacia) affinity
chromatography.
2.12 SDS-PAGE and Western Blot Analysis
The supernatant, the periplasmic extract and cell extract from the
positive clones were separated by reducing and nonreducing
SDS-PAGE. 10% SDS-polyacrylamide slab gel and buffers were prepared
according to Laemmli (Laemmli, 1970). Electrophoresis was performed
at 100V for 45 min After completion of the run, western blot was
carried out as described by Towbin (Towbin et al., 1979). The
nitrocellulose membrane was blocked by 5% nonfat milk in TBS
solution for 1 hour and incubated with anti-idiotype 2E9 antibody
overnight at 4.degree. C. After incubating with HRP-conjugated goat
anti-rat Ig (Fisher Scientific Co., Pittsburgh, Pa.), the signal
was detected by ECL system (Amersham-Pharmacia Biotech).
2.13 Cytotoxicity Assay
Target NMB7 or LAN-1 tumor cells were labeled with
Na.sub.2.sup.51CrO.sub.4 (Amersham Pharmacia) at 100 uCi/10.sup.6
cells at 37.degree. C. for 1 hour. After the cells were washed,
loosely bound .sup.51Cr was leaked for 1 hour at 37.degree. C.
After further washing, 5000 target cells/well were admixed with
lymphocytes to a final volume of 200 .mu.l/well. Antibody dependent
cell-mediated cytotoxicity (ADCC) was assayed in the presence of
increasing concentrations of chimeric antibody. In complement
mediated cytotoxicity (CMC), human complement (at 1:5, 1:15 and
1:45 final dilution) was used instead of lymphocytes. The plates
were incubated at 37.degree. C. for 4 hours. Supernatant was
harvested using harvesting frames (Skatron, Lier, Norway). The
released .sup.51Cr in the supernatant was counted in a universal
gamma-counter (Packard Bioscience, Meriden, Conn.). Percentage of
specific release was calculated using the formula
100%.times.(experimental cpm-background cpm)/(10% SDS releasable
cpm-background cpm), where cpm were counts per minute of .sup.51Cr
released. Total release was assessed by lysis with 10% SDS
(Sigma-Aldrich), and background release was measured in the absence
of cells. The background was usually <30% of total for either
NMB7 or LAN-1 cells. Antibody 3F8 was used as the positive control
(Cheung et al., 1985).
2.14 Iodination
MoAb was reacted for 5 min with .sup.125I (NEN Life Sciences,
Boston, Mass.) and chloramine T (1 mg/ml in 0.3M Phosphate buffer,
pH 7.2) at room temperature. The reaction was terminated by adding
sodium metabisulfite (1 mg/ml in 0.3M Phosphate buffer, pH 7.2) for
2 min Free iodine was removed with A1GX8 resin (BioRad, Richmond,
Calif.) saturated with 1% HSA (New York Blood Center Inc., New
York, N.Y.) in PBS, pH 7.4. Radioactive peak was collected and
radioactivity (mCi/ml) was measured using a radioisotope calibrator
(Squibb, Princeton, N.J.). Iodine incorporation and specific
activities were calculated. Trichloroacetic acid (TCA) (Fisher
Scientific) precipitable activity was generally >90%.
2.15 In Vitro Immunoreactivity of Iodinated Antibody.
Immunoreactivity of radioiodine labeled antibody was assayed using
purified anti-idiotype antibody 2E9 as the antigen. Appropriate
dilutions of .sup.125I labeled antibodies were added to plates in
duplicates, and then transferred to freshly prepared antigen plates
after 1 h and 4 h of binding at 4.degree. C., respectively. The
final binding step was allowed to proceed overnight at 4.degree. C.
The total percent radioactivity bound was a summation of 3 time
points for each antibody dilution. For native 8H9, maximum
immunoreactivity averaged .about.65%, while 8H9 scFv-Fc chimeric
antibody was .about.48%.
2.16 Animal Studies
Athymic nude mice (nu/nu) were purchased from NCI, Frederick Md.
They were xenografted subcutaneously with LAN-1 neuroblastoma cell
line (2.times.10.sup.6 cells/mouse) suspended in 100 ul of Matrigel
(Becton-Dickinson BioSciences, Bedford, Mass.) on the flank. After
3 weeks, mice bearing tumors of 1-1.5 cm in longest dimension were
selected. Animals were injected intravenously (retrorbital plexus)
with 20 .mu.Ci of .sup.125I labeled antibody. They were
anesthetized with ketamine (Fort Dodge Animal Health, Fort Dodge,
Pa.) intraperitoneally and imaged at various time intervals with a
gamma camera (ADAC, Milpitas, Calif.) equipped with grid
collimators. Serial blood samples were collected at 5 min, 1, 2, 4,
8, 18, 24, 48, 72, 120 h from mice injected with 10-11 uCi
.sup.125I labeled antibody. Groups of mice were sacrificed at 24 h,
48 h, and 120 h and samples of blood (cardiac sampling), heart,
lung, liver, kidney, spleen, stomach, adrenal, small bowel, large
bowel, spine, femur, muscle, skin, brain and tumor were weighed and
radioactivity measured by a gamma counter. Results were expressed
as percent injected dose per gram. Animal experiments were carried
out under an IACUC approved protocol, and institutional guidelines
for the proper and humane use of animals in research were
followed.
3. Results
3.1 Anti-8H9-Idiotypic Antibodies
Rat hybridomas specific for 8H9 and nonreactive with control murine
IgG1 were selected. After subcloning by limiting dilution, rat
antibodies were produced by bulk culture in roller bottles and
purified by protein G affinity column By ELISA, 2E9, 1E12, and
1F11, all of rat subclass IgG2a, were specific for 8H9, while
nonreactive with a large panel of purified monoclonal antibodies
(Table I). In contrast, the antibodies 3C2, 4C2 5C7, 7D6 and 8E12
from the same fusions were not specific for 8H9. The rest of the
experiments in this study was carried out using antibody 2E9. 2E9
specifically inhibited the binding of 8H9 to LAN-1 neuroblastoma
(FIG. 18A) and HTB82 rhabdomyosarcoma (FIG. 18B) while control rat
IgG1 (A1G4) had no effect (FIG. 18C).
TABLE-US-00023 TABLE I Anti-8H9-idiotypic antibodies: Specificity
by ELISA 1E12 1F11 3C2 7D6 2E9 MoAb Class .lamda.2a .lamda.2a
.lamda.2b 4C2 .mu. 5C7 .mu. .lamda.1 8E12 .mu. .lamda.2a MOPC 315 a
- - +++ - - - - - 20.4 .lamda.1 - - +++ +++ ++ +++ - - 2C9 .lamda.1
- - +++ +++ +++ +++ ++ - 2E10 .lamda.1 - - +++ - - + - - 3E6
.lamda.1 - - +++ +++ +++ +++ +++ - 3E7 .lamda.1 - - +++ - - + - -
4B6 .lamda.1 - - +++ +++ ++ +++ - - 5F9 .lamda.1 - - +++ +++ +++
+++ + - 8H9 .lamda.1 +++ ++ +++ +++ ++ +++ - ++ MOPC 21 .lamda.1 -
- +++ +++ +++ +++ - - UJ 13A .lamda.1 - - +++ ++ + - - - 3A5
.lamda.2a - - +++ - - - - - HOPC-1 .lamda.2a - - +++ + - - - - 3F8
.lamda.3 - - +++ - - - - - FCOPC21 .lamda.3 - - +++ ++ - ++ - -
NRCO-04 .lamda.3 - - +++ - - - - - R24 .lamda.3 - - +++ - - - - -
TIB114 .lamda.3 - - +++ + - ++ - - Y5606 .lamda.3 - - +++ - - - - -
3A7 .mu. - - + - - - - - 3G6 .mu. - - +++ - - - - - 5F11 .mu. - - +
- - - - - K9 .mu. - - +++ - - - - - MOPC .mu. - - +++ - - - - -
104E Note: OD <0.5 = -, 0.5~1 = +, 1~2 = ++, >2 = +++
3.2 Construction and Expression of 8H9 ScFv
After three rounds of panning on 2E9, the eluted phage was used to
infect E. coli HB2151 cells and scFv expression was induced by
IPTG. ScFv from periplasmic soluble protein fraction was tested for
binding to 2E9 on ELISA. Three 8H9 scFv clones when compared with
the MoAb 8H9 showed similar titers. The clone 8HpHM9F7-1 was
selected for subcloning. The DNA sequence of 8HpHM9F7-1 agreed with
those of the 8H9VH and 8H9VL as well as the CH2-CH3 region of human
gamma chain. The supernatant, periplasmic soluble and cells pellet
lysates of 8HpHM9F7-1 were separated by nonreducing SDS-PAGE, and
analysed by western blotting. A protein band with molecular weight
of 31 KD was found in the supernatant, the periplasmic and cell
pellet extracts using anti-MycTag antibody which recognized the
sequence GAPVPDPLEPR. No such band was detected in control cells or
8HpHM9F7-1 cells without IPTG treatment.
3.3 Construction of Chimeric Mouse scFv-Human Fc
Chimeric clones from CHO and NSO were screened by ELISA binding on
2E9. Clone 1C5 from NSO and clone 1G1 from CHO were chosen for
scale-up production. By SDS-PAGE and by western blot analysis, a
single chain of 54 kD under reducing conditions, and a homodimer of
102 kD under nonreducing conditions were found (FIG. 19). Antigen
specificity was demonstrated by its binding to tumor cells (FIG.
20A, dose titration), and its inhibition by anti-idiotypic antibody
2E9 (FIG. 20B) on FACS analysis.
3.4 In Vitro and In Vivo Properties of scFv-Human Fc
The scFv-Fc chimeric antibody was inefficient in mediating ADCC in
the presence of human lymphocytes or human neutrophils (17% maximum
cytotoxicity at 50:1 E:T ratio compared to >50% by the murine
IgG3 MoAb 3F8). It was also ineffective in CMC (data not shown). In
biodistribution studies, it localized well to HTB82 and LAN-1
xenografts (FIG. 21). Blood clearance studies showed that chimeric
8H9 (102 kD MW) had T-1/2.quadrature. of 5.3 h, and
T-1/2.quadrature. of 43 h when compared to averages of 4.5 h and 71
h, respectively, for native 8H9 (160 kD MW), a result of the
smaller molecular size of the construct (FIG. 22). Similarly,
although the percent injected dose per gram of the chimeric
construct was lower for all tissues (average of 44% at 48 h, and
75% at 120 h), the tumor-non tumor ratios were similar to those of
native 8H9 (98% at 48 h and 85% at 120 h) (Table II).
TABLE-US-00024 TABLE II Percent Injected Dose per gram and
Tumor-non-tumor ratios chimeric native Organs 24 48 120 48 120
Percent Injected dose/gm over time (h) Skin 1.4 0.7 0.2 1.8 0.7
Heart 1.3 0.9 0.4 2.6 0.7 Lung 2.9 1.9 0.5 4.0 1.1 Liver 1.2 0.8
0.2 1.4 0.5 Spleen 0.9 0.5 0.2 1.4 0.4 Kidney 1.5 0.9 0.5 1.9 0.5
Adrenal 0.9 0.5 0.5 1.8 0.3 Stomach 1.3 0.6 0.3 1.3 0.5 Small 0.6
0.3 0.2 0.7 0.2 intestine Large 0.6 0.3 0.2 0.6 0.2 intestine
Bladder 1.2 0.6 0.4 1.0 0.6 Muscle 0.5 0.3 0.2 0.5 0.2 Femur 0.6
0.3 0.2 0.8 0.2 Spine 0.6 0.4 0.2 0.8 0.3 Tumor 4.0 3.6 2.1 9.4 4.0
Brain 0.2 0.1 0.1 0.2 0.1 Blood 5.3 3.1 1.2 8.3 2.3 Tumor: Nontumor
ratios over time (h) Skin 3.0 6.0 10.7 5.2 7.2 Heart 3.3 4.0 5.6
3.6 7.7 Lung 1.6 2.2 4.5 2.3 5.0 Liver 3.5 5.2 8.7 6.5 10.1 Spleen
5.1 8.1 12.8 6.7 15.1 Kidney 2.8 4.3 5.9 5.1 8.9 Adrenal 4.8 8.7
10.0 5.8 11.6 Stomach 3.6 6.7 13.8 7.5 14.5 Small 6.6 11.8 16.0
13.3 21.7 intestine Large 7.1 12.7 25.9 15.7 28.5 intestine Bladder
3.5 14.3 10.2 12.4 12.3 Muscle 7.9 13.6 21.3 18.2 26.8 Femur 6.7
11.8 20.5 11.8 27.9 Spine 6.7 6.8 14.2 11.1 19.6 Tumor 1.0 1.0 1.0
1.0 1.0 Brain 22.7 40.9 38.7 44.6 68.2 Blood 0.8 1.2 1.8 1.1 2.3
##STR00001##
4. Discussion
We demonstrated that by using rat anti-idiotypic antibody as
antigen surrogate, scFv and scFv-fusion proteins can be
conveniently produced. As proof of principle we utilized the
anti-idiotypic antibody to clone scFv from the murine hybridoma
cDNA library. The anti-idiotypic antibody was then used to select
for scFv-Fc chimeric antibodies. Both the scFv and scFv-Fc fusion
protein derived by our method were specific for the natural
antigen, comparable to the native antibody 8H9. However, the
scFv-Fc fusion protein could only mediate ADCC poorly and not CMC
at all.
While scFv provides the building block for scFv-fusion proteins, it
is not the ideal targeting agent by itself. Being a small protein,
its clearance is rapid. Moreover, it is often retained by the
kidney, delivering undesirable side effects if the scFv construct
is cytotoxic. Since avidity is a key parameter in tumor targeting
in vivo, its biggest limitation is its uni-valency and often
suboptimal affinity for the antigen. By using VH-VL linkers of
decreasing length, spontaneous dimeric, trimeric and polymeric scFv
have been produced. However, these oligomers are not bonded by
covalent linkage, and may dissociate in vivo. An alternative
approach is to take advantage of the human Fc, which has the
natural ability to homodimerize through disulfide-bonds, thereby
allowing the juxtaposition of two binding domains. Fc functions
such as CMC and ADCC could also be achieved (Shu et al., 1993; Kato
et al., 1995; Brocks et al., 1997; Wang et al., 1999; Powers et
al., 2001). Unlike standard 2-chain chimeric antibodies, only one
polypeptide is needed for the scFv-Fc chimeric; unbalanced
synthesis of heavy and light chains is not an issue. Larger dimeric
fragments are also likely to have increased serum-half life
compared to scFv and thus improved tumor targeting (Adams et al.,
1993; Wu et al., 1996). Homodimerization of tumor cell-surface
antigens by soluble antibody may also trigger apoptosis of tumor
cells (Ghetie et al., 1997). No less important is the availability
of validated purification techniques using protein A or protein G
through their binding to the Fc portion (Powers et al., 2001).
Tetravalent scFv (monospecific or bispecific) are natural
extensions of the diabody approach to scFv-Fc fusion strategy (Alt
et al., 1999; Santos et al., 1999), where a significant increase in
avidity can be achieved. More recently, scFv-streptavidin fusion
protein has been produced for pretargeted lymphoma therapy (Schultz
et al., 2000). Here scFv-streptavidin forms natural tetramers, to
which biotinylated ligands can bind with high affinity.
Anti-idiotypic antibodies have greatly facilitated clone selection
in the construction of soluble scFv-fusion proteins or cell bound
surface scFv. We have successfully applied similar technology to
anti-GD2 monoclonal antibodies (Cheung et al., 1993). Being
immunoglobulins, their structure, stability, biochemistry, are
generally known. Unlike natural antigens where each individual
system has its unique and difficult to predict properties. As
surrogate antigens, anti-idiotypic antibodies are ideal for
standardization and quality control, especially for initial
clinical investigations where the nature of the antigen is not
fully understood. Potential limitations exist for the anti-idiotype
approach. Only those anti-ids (Ab2.quadrature.) that recognize the
antigen-binding site of the immunizing MoAb can mimic the original
antigen. A reliable test for Ab2.quadrature. is its ability to
induce an antigen-specific immune response. Alternatively, antigen
specificity of the scFv selected by the anti-idiotype must be
validated by binding to cells or membrane preparations. Once
validated, the anti-idiotype can be used as antigen surrogate for
cloning and assay of other scFv-fusion proteins.
Our scFv-Fc fusion protein lacks CMC and ADCC activity. This
finding differs from previous scFv-Fc fusion proteins (Shu et al.,
1993; Wang et al., 1999; Powers et al., 2001). This is unlikely to
be due to the p58 antigen recognized by this scFv, since anti-GD2
scFv-Fc made with the same cassette were also deficient in CMC and
ADCC activity (data not shown). One possible explanation might be
due to the oligosaccharide structures in the Fc region (Wright and
Morrison, 1997). In normal IgG, these oligosaccharides are
generally of complex biantennary type, with low levels of terminal
sialic acid and bisecting N-acetylglucosamine (GlcNAc), the latter
being critical for ADCC. ADCC function is often inefficient among
chimeric antibodies expressed in cell lines which lack the enzyme
.quadrature.(1,4)-N-acetylglucosaminyltransferase III (GnIII)
(Umana et al., 1999), that catalyzes the formation of bisecting
oligosaccharides. This enzyme can be transfected into producer
lines to increase the level of bisecting GlcNAc and to increase the
ADCC function of secreted chimeric antibodies (Umana et al., 1999).
Since our chimeric antibodies from both CHO and NSO expression
systems were inefficient in CMC and ADCC, both cell lines may be
lacking in the GnIII enzyme. It is also possible that the absence
of the CH1 domain in the Fc may modify the accessability of the
ASN297 residue to glycosyltransferases in some scFv-Fc constructs
such as ours (Wright and Morrison, 1997). On the other hand, an
scFv-Fc that lacks binding to Fc receptor may have less nonspecific
binding to white cells, thereby decreasing blood pooling in
targeted therapy. These findings may have implications in scFv-Fc
strategies to improve effector functions.
REFERENCES
Adams, G. P., McGartney, J. E., Tai, M.-S., Oppermann, H., Huston,
J. S., Stafford, W. F., Bookman, M. A., Fand, I., Houston, L. L.
and Weiner, L. W. (1993) Highly specific in vivo tumor targeting by
monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain
Fv. Cancer Research 53, 4026-4034. Alt, M., Muller, R. and
Kontermann, R. E. (1999) Novel tetravalent and bispecific IgG-like
antibody molecules combining single-chain diabodies with the
immunoglobulin y1 Fc or CH3 region. FEBS Letters 454, 90-94. Bird,
R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B.
M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S, and Whitlow,
M. (1988) Single-chain antigen-binding proteins. Science 242,
423-426. Brocks, B., Rode, H. J., Klein, M., Gerlach, E., Dubel,
S., Little, M., Pfizenmaier, K. and Moosmayer, D. (1997) A TNF
receptor antagonistic scFv, which is not secreted in mammalian
cells, is expressed as a soluble mono- and bivalent scFv derivative
in insect cells. Immunotechnology 3, 173-84. Burton, D. R. and
Barbas III, C. G. (1994) Human antibodies from combinatorial
libraries. Advances in Immunology 57, 191-280. Cai, X. and Garen,
A. (1995) Anti-melanoma antibodies from melanoma patients immunized
with genetically modified autologous tumor cells: selection of
specific antibodies from single-chain Fv fusion phage libraries.
Proceedings of the National Academy of Sciences of the United
States of America 92, 6537-41. Cheung, N. K., Canete, A., Cheung,
I. Y., Ye, J. N. and Liu, C. (1993) Disialoganglioside GD2
anti-idiotypic monoclonal antibodies. International Journal of
Cancer 54, 499-505. Cheung, N. K., Saarinen, U., Neely, J.,
Landmeier, B., Donovan, D. and Coccia, P. (1985) Monoclonal
antibodies to a glycolipid antigen on human neuroblastoma cells.
Cancer Research 45, 2642-2649. DeNardo, S. J., DeNardo, G. L.,
DeNardo, D. G., Xiong, C. Y., Shi, X. B., Winthrop, M. D., Kroger,
L. A. and Carter, P. (1999) Antibody phage libraries for the next
generation of tumor targeting radioimmunotherapeutics. Clinical
Cancer Research 5, 3213s-3218s. Eshhar, Z., Waks, T., Gross, G. and
Schindler, D. G. (1993) Specific activation and targeting of
cytotoxic lymphocytes through chimeric single chains consisting of
antibody-binding domains and the gamma or zeta subunits of the
immunoglobulin and T-cell receptors. Proceedings of the National
Academy of Sciences of the United States of America 90, 720-4.
George, A. J. T., Spooner, R. A. and Epenetos, A. A. (1994)
Applications of Monoclonal Antibodies in Clinical Oncology.
Immunology Today 15, 559-561. Ghetie, M. A., Podar, E. M., Ilgen,
A., Gordon, B. E., Uhr, J. W. and Vitetta, E. S. (1997)
Homodimerization of tumor-reactive monoclonal antibodies markedly
increases their ability to induce growth arrest or apoptosis of
tumor cells. Proceedings of the National Academy of Sciences of the
United States of America 94, 7509-14. Huston, J. S., Levinson, D.,
Mudgett-Hunter, M., Tai, M. S., Novotny, J., Margolies, M. N.,
Ridge, R. J., Bruccoleri, R. E., Haber, E. and Crea, R. (1988)
Protein engineering of antibody binding sites: recovery of specific
activity in an anti-digoxin single-chain Fv analogue produced in
Escherichia coli. Proceedings of the National Academy of Sciences
of the United States of America 85, 5879-83. Kato, T., Sato, K.,
Suzuki, S., Sasakawa, H., Kurokawa, M., Nishioka, K. and Yamamoto,
K. (1995) Mammalian expression of single chain variable region
fragments dimerized by Fc regions. Molecular Biology Reports 21,
141-146. Kipriyanov, S. M., Bretling, F., Little, M. and Dubel, S.
(1995) Single-chain antibody streptavidin fusions: tetrameric
bifunctional scFv-complexes with biotin binding activity and
enhanced affinity to antigen. Human Antibodies Hybridomas 6,
93-101. Laemmli, U. K. (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 227,
680-85. Lu, J. and Sloan, S. R. (1999) An alternating selection
strategy for cloning phage display antibodies. Journal of
Immunological Methods 228, 109-119. Michael, N. P., Chester, K. A.,
Melton, R. G., Robson, L., Nicholas, W., Boden, J. A., Pedley, R.
B., Begent, R. H., Sherwood, R. F. and Minton, N. P. (1996) In
vitro and in vivo characterisation of a recombinant
carboxypeptidase G2::anti-CEA scFv fusion protein. Immunotechnology
2, 47-57. Modak, S., Kramer, K., Humayun, G., Guo, H. F. and
Cheung, N. K. V. (2001) Monoclonal antibody 8H9 targets a novel
cell surface antigen expressed by a wide spectrum of human solid
tumors. Cancer Research 61, 4048-4054. Powers, D. B., Amersdorfer,
P., Poul, M. A., Nielsen, U. B., Shalaby, R., Adams, G. P., Weiner,
L. M. and Marks, J. D. (2001) Expression of single-chain Fv-Fc
fusions in pinchia pastoris. Journal of Immunological Methods 251,
123-135. Raag, R. and Whitlow, M. (1995) Single-chain Fvs. FASEB
Journal 9, 73-80. Santos, A. D., Kashmiri, S. V., Hand, P. H.,
Schlom, J. and Padlan, E. A. (1999) Generation and characterization
of a single gene-encoded single-chain-tetravalent antitumor
antibody. Clinical Cancer Research 5, 3118s-3123s. Schultz, J.,
Lin, Y., Sanderson, J., Zuo, Y., Stone, D., Mallett, R., Wilbert,
S, and Axworthy, D. (2000) A tetravalent single-chain
antibody-streptavidin fusion protein for pretargeted lymphoma
therapy. Cancer Research 60, 6663-6669. Shu, L., Qi, C. F., Schlom,
J. and Kashmiri, S. V. (1993) Secretion of a single-gene-encoded
immunoglobulin from myeloma cells. Proceedings of the National
Academy of Sciences of the United States of America 90, 7995-9.
Thanavala, Y. M., Brown, S. E., Howard, C. R., Roitt, I. M. and
Steward, M. W. (1986) A surrogate hepatitis B virus antigenic
epitope represented by a synthetic peptide and an internal image
antiidiotype antibody. Journal of Experimental Medicine 164,
227-236. Towbin, H., Staehelin, T. and Gordon, J. (1979)
Electrophoretic transfer of proteins from polyacrylamide gels to
nitrocellulose sheets: procedure and some applications. Proceedings
of the National Academy of Sciences of the United States of America
76, 4350-4. Tur, M. K., Huhn, M., Sasse, S., Engert, A. and Barth,
S. (2001) Selection of scFv phages on intact cells under low pH
conditions leads to a significant loss of insert-free phages.
Biotechniques 30, 404-413. Umana, P., Jean-Mairet, J., Moudry, R.,
Amstutz, H. and Bailey, J. E. (1999) Engineered glycoforms of an
antineuroblastoma IgG1 with optimized antibody-dependent cellular
cytotoxic activity. Nature Biotechnology 17, 176-180. Wagner, U.,
Schlebusch, H., Kohler, S., Schmolling, J., Grunn, U. and Krebs, D.
(1997) Immunological responses to the tumor-associated antigen
CA125 in patients with advanced ovarian cancer induced by the
murine monoclonal anti-idiotype vaccine ACA125. Hybridoma 16,
33-40. Wang, B., Chen, Y. B., Ayalon, O., Bender, J. and Garen, A.
(1999) Human single-chain Fv immunoconjugates targeted to a
melanoma-associated chondroitin sulfate proteoglycan mediate
specific lysis of human melanoma cells by natural killer cells and
complement. Proceedings of the National Academy of Sciences of the
United States of America 96, 1627-32. Watters, J. M., Telleman, P.
and Junghans, R. P. (1997) An optimized method for cell-based phage
display panning. Immunotechnology 3, 21-9. Wikstrand, C. J., Hale,
L. P., Batra, S. K., Hill, M. L., Humphrey, P. A., Kurpad, S. N.,
McLendon, R. E., Moscatello, D., Pegram, C. N. and Reist, C. J.
(1995) Monoclonal antibodies against EGFRvIII are tumor specific
and react with breast and lung carcinomas and malignant gliomas.
Cancer Research 55, 3140-8. Winter, G., Griffiths, A. D., Hawkins,
R. E. and Hoogenboom, H. R. (1994) Making antibodies by phage
display technology. Annual Review of Immunology 12, 433-55. Winter,
G. and Milstein, C. (1991) Man-made antibodies. Nature 349,
293-299. Wright, A. and Morrison, S. L. (1997) Effect of
glycosylation on antibody function: implications for genetic
engineering. Trends in Biotechnology 15, 26-31. Wu, A. M., Chen,
W., Raubitschek, A., Williams, L. E., Neumaier, M., Fischer, R.,
Hu, S. Z., Odom-Maryon, T., Wong, J. Y. and Shively, J. E. (1996)
Tumor localization of anti-CEA single-chain Fvs: improved targeting
by non-covalent dimers Immunotechnology 2, 21-36.
Sixth Series of Experiments
Using Anti-Idiotypic Antibody to Enhance scFv Chimeric Immune
Receptor Gene Transduction and Clonal Expansion of Human
Lymphocytes
Background: Chimeric immune receptors (CIR) transduced into
lymphocytes link target recognition by single chain antibody Fv
(scFv) to activation through CD28/TCR. signaling. The murine
monoclonal antibody (MoAb) 8H9 reacts with a novel antigen widely
expressed on solid tumors (Cancer Research 61:4048, 2001). We want
to test if its anti-idiotypic MoAb 2E9 can optimize the CIR
technology.
Methods: Rat anti-idiotypic MoAb 2E9 (IgG2a) was used as an antigen
surrogate for initial cloning of 8H9scFv from the hybridoma cDNA
library. A CIR consisting of human CD8-leader sequence, 8H9scFv,
CD28 (transmembrane and cytoplasmic domains), and TCR-zeta chain
was constructed, ligated into the pMSCVneo vector, and used to
transfect the packaging line GP+envAM12 bearing an amphotropic
envelope.
Results: Three sequential affinity enrichments with MoAb 2E9
significantly improved the percentage of producer clones positive
for surface 8H9-scFv and the efficiency of their supernatant in
transducing the indicator cell line K562. By three weeks of in
vitro culture, >95% of transduced primary human lymphocytes were
CIR-positive. With periodic stimulation with soluble 2E9, these
lymphocytes underwent "monoclonal" expansion, reaching 50-100 fold
increase by 2 months. They mediated antigen-specific non-MHC
restricted cytotoxicity efficiently. When injected intravenously,
they inhibited tumor growth in SCID mice xenografted with
rhabdomyosarcoma.
Conclusion: Anti-idiotypic antibody may provide a useful tool,
especially for carbohydrate or unstable antigens, in facilitating
the cloning of scFv and their CIR fusion constructs, as well as
their transduction into human lymphocytes.
Introduction
Adoptive cell therapy using ex vivo expanded tumor-selective
T-cells can effect dramatic remissions of virally induced
malignancies, a process critically dependent on clonal frequency,
where rapid exponential expansion of specific cytolytic
T-lymphocytes (CTL) is required. T-cells proliferate when activated
(e.g. anti-CD3) but apoptose unless a costimulatory signal (e.g.
anti-CD28) is provided (1). However, human tumor targets often lack
costimulatory molecules (e.g. CD80), or overstimulate inhibitory
receptors (e.g. CTL4) such that the CD28 pathway is derailed. In
addition, many tumors down-regulate major histocompatibility
complex (MHC) molecules to escape engagement by the T-cell receptor
(TCR). Through genetic engineering, chimeric immune receptors (CIR)
linking tumor-selective scFv to T-cell signal transduction
molecules (e.g. TCR-zeta chain and CD28) will activate lymphocytes
following tumor recognition, triggering the production of cytokines
and tumor lysis (2-7). T-cell can also be genetically engineered to
secrete cytotoxic cytokines (8), toxins (9) or to metabolize
prodrugs (10.mu., 11). However, significant technologic gaps
remain: (1) Gene transduction into human lymphocytes is
inefficient, (2) antigen specific T-cells cannot be easily enriched
and expanded, and (3) optimal T-cell activation may require
multiple signals. Furthermore, although CIR redirected T-cells can
recycle their lytic activity (12), a costimulatory signal, either
through CD28 or 4-1BB engagement, may help reduce
activation-induced apoptotic death. CIR with multidomains was
recently described, where the intracellular domain of CD28 was
ligated to the 5' end of TCR zeta chain and introduced into Jurkat
cells, with the expected "two birds with one stone effect" when
scFv binds to tumor cells (13). IL-2 production was 20 times more
than CIR with zeta chain only. Whether this same effect can be
achieved with primary human T-cells is not known.
To monitor scFv gene expression, anti-linker antibody may be
useful, although its efficiency depends on the accessibility of the
scFv-linker portion. Although purified antigens can also be used to
monitor scFv expression, certain classes (complex carbohydrates or
unstable antigens) can be difficult to prepare and their chemistry
highly variable. Without a standardized reagent for affinity
purification or enrichment of virus producer cells, monitoring and
sorting of transduced lymphocytes, CIR technology remains
inefficient. Recently Eshhar et al described a dicistronic
construct consisting of scFv-CD28-(and green fluorescent protein
(GFP), where the latter was used to monitor gene transduction and
to enrich producer lines (7). Although GFP can validate the gene
transfer process, its added immunogenicity and its safety in
clinical applications remain uncertain.
Anti-idiotypic antibodies are frequently used as antigen-mimics for
infectious diseases and cancer (14, 15). Internal image rat
anti-idiotypic antibodies can be conveniently produced against
mouse MoAb. Since large scale production of clinical grade MoAb is
now routine, anti-idiotypic antibodies may be ideal surrogates
especially if the antigen is not easily available. In addition, the
biochemistry of immunoglobulins in positive selection (panning,
affinity chromatography, sorting) and binding assays is well-known
and is easy to standardize. We recently described a novel tumor
antigen reactive with a murine MoAb 8H9 (16). The antigen was
difficult to purify given its lability and glycosylation. Here we
demonstrate that anti-idiotypic MoAb can be used as surrogate
antigens for cloning CIR into lymphocytes, i.e. a CIR of 8H9scFv,
human CD28 and human TCR-zeta chain. Anti-idiotypic MoAb allows
rapid affinity enrichment of producer cell line, monitoring of scFv
expression on cells, and in vitro clonal expansion of transduced
lymphocytes. Highly cytotoxic lymphocytes, both in vitro and in
vivo, can be produced in bulk. Besides providing an antigen
surrogate, anti-idiotypic MoAb appears to have utility for the
optimization and quality control of scFv-based gene therapies.
Materials and Methods
Materials. Cells were cultured in RPMI 1640 with 10% newborn calf
serum (Hyclone, Logan, Utah) supplemented with 2 mM glutamine, 100
U/ml of penicillin and 100 ug/ml of streptomycin. The BALB/c
myeloma proteins, MOPC-104E, TEPC-183, MOPC-351, TEPC-15, MOPC-21,
UPC-10, MOPC-141, FLOPC-21, Y5606, were purchased from
Sigma-Aldrich Co., St. Louis, Mo. MoAb R24, V1-R24, and K9 were
gifts from Dr. A. Houghton, OKB7 and M195 from Dr. D. Scheinberg,
and 10-11 (anti-GM2) from Dr. P. Livingston of Memorial
Sloan-Kettering Cancer Center, New York; 528 from Dr. J. Mendelsohn
(MD Anderson Cancer Center, Houston, Tex.). 2E6 (rat anti-mouse
IgG3) was obtained from hybridomas purchased from ATCC (Rockville,
Md.). NR-Co-04 was provided by Genetics Institute (Cambridge,
Mass.). LS2D173 (anti-GM2) was provided by Dr. L. Grauer
(Hybritech, CA). From our laboratory, 3F8 was an IgG3 MoAb specific
for ganglioside GD2 (17); 5F9, 8H9, 3A5, 3E7, 1D7, 1A7 were
produced against human neuroblastoma, 2C9, 2E10 and 3E6 against
human breast carcinoma: 4B6 against glioblastoma multiforme. They
were all purified by protein A or protein G (Pharmacia, Piscataway,
N.J.) affinity chromatography.
Anti-8H9-idiotypic MoAb. Anti-idiotypic antibodies were produced
from LOU/CN rats as previously described (18). Clones were selected
based on selective binding to 5F11 antibody and not to other
myelomas. Repeated subcloning was done using limiting dilution
until the cell lines became stable. Among the three specific rat
IgG2a clones (2E9, 1E12, 1F11), 2E9 was chosen for scaled up
production using high density miniPERM bioreactor (Unisyn
technologies, Hopkinton, Mass.), and purified by protein G affinity
chromatography (Hitrap G, Amersham-Pharmacia, Piscataway, N.J.).
The IgG fraction was eluted with pH 2.7 glycine-HCl buffer and
neutralized with 1 M Tris buffer pH 9. After dialysis in PBS at
4.degree. C. for 18 hours, the purified antibody was filtered
through a 0.2 um Millipore filter (Millipore Inc. Bedford Mass.),
and stored frozen at -70.degree. C. Purity was determined by
SDS-PAGE electrophoresis using 7.5% acrylamide gel. ELISA was used
to detect rat anti-idiotypic antibodies (Ab2) as previously
described (18). Rat IgG1 anti-5F11 anti-idiotypic MoAb was
similarly produced.
Construction of ScFv Gene scFv was constructed from 8H9 hybridoma
cDNA by recombinant phage display using a scFv construction kit
according to manufacturer's instructions with modifications
(Amersham-Pharmacia). Amplified ScFv DNA was purified by glassmilk
beads and digested with Sfi I and Not I restriction endonucleases.
The purified scFv of 8H9 was inserted into the pHEN1 vector (kindly
provided by Dr. G. Winter, Medical Research Council Centre,
Cambridge, UK) containing SfiI/NcoI and Not I restriction sites.
Competent El Coli XL 10Blue cells (Stratagene, La Jolla, Calif.)
were transformed with the pHEN1 phagemid. Helper phage M13 KO7
(Pharmacia) was added to rescue the recombinant phagemid. The
phagemid 8HpHM9F7-1 was chosen for the rest of the experiments. The
supernatant, the periplasmic extract and cell extract from the
positive clones separated by nonreducing SDS-PAGE and western
blotting (19) using anti-Myc Tag antibody demonstrated a 31 kD
band.
Enrichment of Recombinant Phagemid by Panning 50 ul of anti-8H9
idiotype antibody 2E9 (50 ug/ml) in PBS were coated on the 96-well
polyvinyl microtiter plates and incubated at 37.degree. C. for 1
hour. 100 ul of the supernatant from phage library were added to
each well and incubated for 2 hours. The plate was washed 10 times
with PBS containing 0.05% BSA. Antigen-positive recombinant phage
captured by the idiotype 2E9 was eluted with 0.1M HCl (pH 2.2 with
solid glycine and 0.1% BSA) and neutralized with 2M Tris solution.
This panning procedure was repeated three times.
ELISA The selected phage was used to reinfect E. coli XL 1-Blue
cells. Colonies were grown in 2.times.YT medium containing
ampicillin (100 ug/ml) and 1% glucose at 30.degree. C. until the
optical density at 600 nm of 0.5 was obtained. Expression of scFv
antibody was induced by change of the medium containing 100 uM IPTG
(Sigma-Aldrich) and incubating at 30.degree. C. overnight. The
supernatant obtained from the medium by centrifugation was directly
added to the plate coated with idiotype 2E9. The pellet was
resuspended in the PBS containing 1 mM EDTA and incubated on ice
for 10 min. The periplasmic soluble antibody was collected by
centrifugation again and added to the plate. After incubating 2
hours at 37.degree. C., plates were washed and anti-MycTag antibody
(clone 9E10 from ATCC) was added to react for 1 hour at 37.degree.
C. After washing, affinity purified goat anti-mouse antibody
(Jackson Immunoresearch, West Grove, Pa.) was allowed to react for
1 hour at 37.degree. C. and the plates were developed with the
substrate o-phenylenediamine (Sigma-Aldrich).
Construction of sc8H9-hCD28.sub.TM-hCD28.sub.cyto-hTCRzeta-pMSCVneo
Using the assembled gene sequences, secondary PCR amplifications
using synthetic oligodeoxynucleotide primers (see below) were
performed. Briefly, a 50 .mu.l reaction mixture containing 200
.mu.M of each deoxynucleotide triphosphate, 0.2 .mu.M of each
primer, 2 units of AmpliTag Gold DNA polymerase (Applied
Biosystems, Foster City, Calif.), and 50 ng of template DNA was
subjected to a 10 min denaturation and activation step at
95.degree. C., followed by 30 cycles of denaturation (1 min at
95.degree. C.), annealing (2 min at 55.degree. C.), and extension
(2 min at 72.degree. C.). This was followed by a final extension
for 8 min at 72.degree. C. Each of the amplified products was
purified with Geneclean Kit (Bio 101, Vista, Calif.).
Synthetic Oligodeoxynucleotide Primers for DNA Amplification
TABLE-US-00025 hCD8a leader-scFv-CD28: 355 S Sense Primer (Hpa
I-Human CD8a Leader) 5'-TTA TTA CGA GTT/AAC ATG GCC TTA CCA SEQ ID
NOs: 15-16 GTG ACC-3'; 355 A Antisense Primer (Xho I-Human CD28)
5'-CTT GGT C/TCGAG TGT CAG GAG CGA TAG SEQ ID NOs: 17-18 GCT GC-3';
scFv8H9: 365 S Sense Primer (Cla I-8H9 heavy chain) 5'-TTA TTA CGA
AT/CGAT T GCC CAG GTC AAA SEQ ID NOs: 19-20 CTG-3'; 365 A Antisense
Primer (Not I-8H9 light chain) 5'-CTT GGT G/CGGCCGC CTG TTT CAG CTC
SEQ ID NOs: 21-22 CAG-3'; hTCR-zeta chain 379S Sense primer (Bst U
I-CD28 end-Xho I-hTCR zeta [cytoplasmic domain]) 5'-CG/C GAC TTA
GCA SEQ ID NOs: 23-26 GCC TAT CGC TCC TGg CAC/TCG AGa AGA GTG AAG
TTC-3'; 379A Antisense Primer (Bg1II-hTCR z) 5'-CTT GGT SEQ ID NOs:
27-28 A/GA TCT TCA GCG AGG GGG CAG GGC-3'.
Templates for DNA Amplification and Construction The single gene
encoding hCD8a-leader-sc3G6-CD28 was previously described (20). Its
cDNA was generated by PCR using the Hpa I, Xho I fragment of
hCD8a-leader-scFv-CD28 cDNA, and ligated into pMSCVneo vector
(Clontech, Palo Alto, Calif.). ScFv-8H9 was amplified from the
8HpHM9F7-1 phagemid. Excised 8H9 scFv gene was then swapped into
the hCD8a-leader-scFv3G6-CD28 cassette of pMSCVneo using the Cla
I-Not I restriction enzymes. Human TCR-zeta-chain was amplified
from the plasmid pcDNA3.1/VJABLZH (kindly provided by Dr. Ira
Bergman, University of Pittsburgh, Pa.), and ligated downstream of
CD28 gene, using Xho I and Bgl II restriction sites. Using the
method supplied by manufacturer (Stratagene), competent E. coli XL
1-Blue cells were transformed with the vector pMSCVneo containing
the insert. All gene constructs were checked by DNA sequencing.
Cell Culture and Transfection The amphotropic packaging cell line
GP+envAM12 and all retroviral producer lines were maintained in
Dulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, Md.)
supplemented with glutamine, penicillin, streptomycin (Gibco-BRL),
and 10% fetal bovine serum (Gibco-BRL). Using effectene
transfection Reagent (Qiagen, Valencia, Calif.), recombinant
retrovirus was produced by the transfection of vector DNA into
GP+envAM12 packaging cells (kindly provided by Genetix
Pharmaceuticals, Cambridge, Mass.). Cells were fed every 3 days
with G418 (400 ug/ml; Gibco-BRL). Resistant clones were selected
after a 10-day period.
Enrichment and Cloning of Packing Lines by Affinity Column The
retroviral producer lines were affinity enriched using MACS goat
anti-rat IgG MicroBeads on the MiniMACS system (Miltenyi, Auburn,
Calif.). In brief, the transduced packing lines were reacted with
purified rat anti-idiotypic antibodies (10 ug per 10.sup.6 packing
cells) on ice for 30 minutes, washed and then applied to the
anti-rat column. Cell were eluted according to manufacturer's
instructions and recultured at 37.degree. C. for 24 hours.
Following staining with anti-idiotypic antibody 2E9 or 1E12,
immunofluorescence was detected with FITC conjugated mouse anti-rat
IgG antibody and analyzed by a FACSCalibur flow cytometer (Becton
Dickinson Immunocytometry systems, San Jose, Calif.). A series of
three affinity purifications is performed on the retroviral
producer line before subcloning by limiting dilution.
Virus-containing supernatant from each clone was used to infect
K562 cells, and gene transduction was measured by surface
expression of scFv on K562 using FACS. One of the scFv-transduced
K562 cell lines was further enriched by MACS system before cloning
by limiting dilution.
Peripheral Blood Mononuclear cells (PBMCs) PBMCs were isolated by
centrifugation on Ficoll (density, 1.077 g/ml) for 30 min at
25.degree. C. and washed twice with PBS. They were activated with
soluble anti-CD3 (1 .mu.g/ml; clone OKT3; PharMingen, San Diego,
Calif.) and anti-CD28 (1 ug/ml; clone CD28.2; PharMingen) MoAbs for
3 days at 37.degree. C. In some experiments, immobilized anti-CD3
and anti-CD28 MoAbs were used, where 12-well non-tissue
culture-treated plates were incubated with the antibody (1 .mu.g/ml
in PBS) at 1 ml/well for 4 hours at 37.degree. C. The coated plates
were blocked with 1% HSA in PBS for 30 min at room temperature,
washed once with PBS, and then used for PBMC activation. PBMCs
(10.sup.6/ml) were cultured in RPMI 1640 supplemented with 10%
human AB serum (Gemini Bio-Products, Woodland, Calif.), 50 .mu.M
2-mercaptoethanol, 2 .mu.M L-glutamine, and 1%
penicillin-streptomycin (Gibco-BRL), for a total of 3 days before
retroviral transfection.
Retroviral Transduction Protocol The target cells (e.g. K562 or
cultured PBMCs) were resuspended at a concentration of
1-5.times.10.sup.5 cells/ml of freshly harvested supernatant from
retroviral producer cells, containing 8-10 ug/ml hexadimethrine
bromide (polybrene, Sigma), centrifuged at 1000.times.g at room
temperature for 60 minutes, and then cultured in 12-well tissue
culture plates overnight. The viral supernatant was then aspirated
and fresh IMDM (Gibco) medium containing 100 U/ml of IL2 and
changed approximately every 5 days to maintain a cell count between
1-2.times.10.sup.6 cells/ml (21). After 2 weeks in culture, soluble
anti-idiotypic antibody 2E9 was added at 3-10 ug/ml to the
transfected lymphocytes for 3 days out of every 2-week culture
period, to ensure clonal expansion of the scFv-positive transfected
lymphocytes.
Cytotoxicity Assay Neuroblastoma targets NMB-7 and LAN-1 or
rhabdomyosarcoma HTB-82 tumor cells were labeled with
Na.sub.2.sup.51CrO.sub.4 (Amersham Pharmacia Biotechnology Inc.,
Piscataway, N.J.) at 100 uCi/10.sup.6 cells at 37.degree. C. for 1
hour. After the cells were washed, loosely bound .sup.51Cr was
removed by washing. 5000 target cells/well were admixed with
lymphocytes to a final volume of 200 .mu.l/well. Following a 3
minute centrifugation at 200.times.g, the plates were incubated at
37.degree. C. for 4 hours. Supernatant was harvested using
harvesting frames (Skatron, Lier, Norway). The released .sup.51Cr
in the supernatant was counted in a universal gamma-counter
(Packard Bioscience, Meriden, Conn.). Percentage of specific
release was calculated using the formula 100%.times.(experimental
cpm-background cpm)/(10% SDS releasable cpm-background cpm), where
cpm are counts per minute of .sup.51Cr released. Total release was
assessed by lysis with 10% SDS (Sigma-Aldrich), and background
release was measured in the absence of cells. The background was
usually <30% of total for these cell lines.
Mice and Treatment CB-17 SCID-Beige mice were purchased from
Taconic (Germantown, N.Y.). Tumor cells were planted
(2.times.10.sup.6 cells) in 100 ul of Matrigel (BD BioSciences,
Bedford, Mass.) subcutaneously. Following implantation, tumor sizes
(maximal orthogonal diameters) were measured. Tumor volume was
calculated as 4Br.sup.3/3 where r is the mean tumor radius.
Treatment studies started in groups of 5 mice per cage when tumor
diameter reached 0.8 cm, usually by one week of tumor implantation.
Mice received 5 weekly intravenous lymphocyte injections by
retroorbital route, 2.times.10.sup.6 per injection together with
500 U of IL-2 ip. 50 ug of anti-idiotypic antibody was administered
ip 3 days after each lymphocyte injection. Tumor sizes were
measured twice a week. Experiments were carried out under an IACUC
approved protocol and institutional guidelines for the proper, and
humane use of animals in research were followed.
Statistical Analysis Tumor growth was calculated by fitting a
regression slope for each individual mouse to log transformed
values of tumor size. Mean slope scores were back-transformed to
give an estimate of the percent increase in tumor size per day.
Slopes were compared between groups.
Results
Anti-8H9-idiotypic antibodies Rat hybridomas specific for 8H9 and
nonreactive with control murine MoAb (IgM, IgG1 and other
subclasses) were selected. By ELISA, 2E9, 1E12, and 1F11 were all
of rat subclass IgG2a. The antibody 2E9 was chosen for the rest of
the experiments.
Construction and expression of 8H9 scFv After secondary PCR
amplification, the PCR product of scFv fitted with Sfi I and Not I
restriction sites were inserted into pHEN1 vectors. Three rounds of
panning were conducted to enrich for 2E9-binding recombinant
phages. The phages eluted from the third round panning were used to
infect E. coli HB2151 cells and induced by IPTG for expression.
ScFv periplasmic soluble protein was allowed to react in plates
coated with 2.5 ug 2E9/well and assayed by ELISA as described in
Material and Methods. The clone 8HpHM9F7-1 was selected for
subcloning. The scFv DNA sequence of 8HpHM9F7-1 agreed with those
of the VH and VL regions of the MoAb 8H9. The supernatant,
periplasmic soluble and cells pellet lysates of 8HpHM9F7-1 were
separated by nonreducing SDS-PAGE, and analyzed by western
blotting. A protein band with the apparent molecular weight of 31
KD was found in the supernatant, the periplasmic and cell pellet
extracts using anti-MycTag antibody which recognized the sequence
GAPVPDPLEPR. No such band was detected in control cells or
8HpHM9F7-1 cells without IPTG treatment.
Construction of sc8H9-CD28-hTCRzeta-pMSCVneo Using the assembled
gene sequences, secondary PCR amplifications using synthetic
oligodeoxynucleotide primers were performed using synthetic
oligodeoxynucleotide primers 355S, 355A for the hCD8a
leader--scFv--CD28, 365S, 365A for scFv8H9, and 379S, 379A for
hTCR-zeta chain. The final gene construction
hCD8_leader-8H9scFv-hCD28.sub.TM-hCD28.sub.cyto-TCR. was
transfected into the amphotropic packaging line GP+envAM12, and
selected in G418.
Enrichment and cloning of packing lines by affinity column The
retroviral producer lines were affinity-enriched using MACS goat
anti-rat IgG MicroBeads on the MiniMACS system. Following each
enrichment, viral supernatant from the producer line was used to
infect the erythroleukemia line K562. Surface 8H9-scFv expression
on both the producer lines and the transfected K562 (3-5 days after
infection) were measured by immunofluorescence using anti-idiotypic
antibody 2E9. With each successive affinity enrichment (FIGS. 23A
and 23C) of producer line and subsequent successive subcloning
(FIGS. 1B and 1D), the surface expression (mean fluorescence) of
8H9-scFv increased and became more homogeneous for the producer
clones (FIGS. 23A and 23B) and for the indicator line K562 (FIGS.
23C and 23D).
Retroviral transduction of primary human peripheral blood
mononuclear cells Following activation in vitro with soluble
anti-CD3 and anti-CD28, primary human peripheral blood mononuclear
cells were infected with the virus from producer line supernatant
by centrifugation at 1000.times.g for 60 minutes at room
temperature. By 21 days of in vitro culture, close to 100% of cells
were scFv-positive by FACS (FIG. 24). This clonal evolution to
homogeneity was found in CD4+, CD8+ and the small CD56+
populations. Soluble anti-idiotypic MoAb 2E9 was added at 3-10
ug/ml to the transfected lymphocytes for 3 days out of every 2
weeks, to stimulate clonal expansion of the scFv-positive
transfected lymphocytes (FIG. 25). ScFv expression was constant
throughout until at least day 62 (FIG. 24), while the cells
underwent active clonal expansion of 100-fold. The proportion of
CD8+ cells increased steadily from an initial 20-60% to 90% by day
40 of culture.
Transduced lymphocytes carried out efficient non MHC-restricted
cytotoxicity in vitro against neuroblastoma and rhabdomyosarcoma In
vitro cytotoxicity against NMB-7 (FIG. 26A) and LAN-1 (FIG. 26B)
neuroblastoma, or rhabdomyosarcoma HTB-82 (FIG. 26C) were
efficient, all inhibitable by 8H9 antibody demonstrating antigen
specificity. Daudi cell line (FIG. 26D) was not killed because it
was antigen-negative. This cytotoxicity was independent of target
HLA expression or HLA types. Unmodified lymphocytes from the same
donor, cultured under the same conditions (100 U/ml of IL2), did
not show antigen-specific killing (LAK, FIG. 26).
Inhibition of rhabdomyosarcoma tumor xenografted in SCID mice.
Human rhabdomyosarcoma was strongly reactive with 8H9, but not with
5F11 (anti-GD2) antibodies. To study the in vivo effects of
8H9scFv-CIR gene-modified lymphocytes, we used 5F11scFv-CIR as
control. 5F11scFv-CIR modified lymphocytes could kill tumors in
vitro, but only if they were GD2-positive (data not shown). When
subcutaneous tumor implants grew to 0.8 cm diameter, mice were
treated with 2.times.10.sup.6 gene-modified human lymphocytes
intravenously plus 500 U of IL2 intraperitoneally once a week for a
total of 5 weeks. 50 ug of anti-idiotypic antibody 2E9 was given ip
3 days after each lymphocyte infusion. All groups received IL2.
Control groups received either no cells+2E9, cultured unmodified
lymphocytes+2E9 (LAK), or 5F11scFv-CIR modified
lymphocytes+anti-idiotype 1G8 (specific for 5F11 idiotype).
Suppression of tumor growth was most significant with lymphocytes
transduced with the 8H9scFv-CIR gene (p=0.066, FIG. 27). Although
5F11scFv-CIR modified lymphocytes also delayed tumor growth, they
were not different from unmodified lymphocytes.
Discussion
The use of retroviral vectors to transduce chimeric immune
receptors into primary human lymphocytes has been limited by the
low gene transfer efficiency when viral supernatant infections were
carried out. Transfer rates into primary human T cells using
amphotropic virus ranged from 1 to 12% (22). Several strategies
were explored to increase the transduction rates to 20-50%. These
include: (1) using gibbon ape leukemia virus (GaLV strain SEATO)
pseudotyped virions (20, 23, 24), (2) coculturing producer and
target cells (25) where the clinical safety was of some concern,
(3) using phosphate depletion followed by centrifugation and
incubation at 32.degree. C. (22), (4) adding fibronectin CH296 to
enhance virus/lymphocyte interactions (26). More recently, Eshhar
et al described a dicistronic construct consisting of
scFv-CD28-(and green fluorescent protein (GFP), where the latter
was used to monitor gene transduction and to enrich producer line
(7). In our study, we used anti-idiotypic antibody to select for
high surface scFv-expressing producer lines with improved
efficiency of gene transduction. More importantly, lymphocytes
transduced by CD-28-chimeric fusion receptors proliferated in the
presence of the anti-idiotypic MoAb to become "monoclonal" with
respect to scFv expression, in both the CD4+ and CD8+ populations.
These lymphocytes possessed antigen-specific tumoricidal activity
both in vitro and in vivo that was non-MHC restricted. Whether
CD56-positive cells (presumably NK cells) acquire similar abilities
will need further studies, although activation of NK cells through
CD28 signaling has been reported previously (27).
We have shown that anti-idiotypic antibodies can facilitate clone
selection in the construction of soluble scFv-fusion proteins or
cell bound surface scFv. We have successfully applied similar
technology to the GD2 antigen system (unpublished data). Being
immunoglobulins, their structure, stability, biochemistry are
generally known. This is in contrast to natural antigens where each
individual system has its unique and often difficult-to-predict
properties. As surrogate antigens, anti-idiotypic MoAb are ideal
for standardization and quality control, especially for initial
clinical investigations of carbohydrate antigens or when the nature
of the antigen is not fully understood.
The advantage of using anti-idiotypic antibody for affinity
purification and for clonal expansion of gene-modified lymphocytes
are many fold. To prepare polyclonal CTLs specific for a tumor
target, lymphocytes have to be pulsed periodically in vitro with
the tumor cells (21). Clearly this can create safety (tumor
contamination) and quality control issues. In contrast,
anti-idiotypic MoAb can be manufactured under standard good
manufacturing practice (GMP) conditions, with ease of manipulation
both in vitro or in vivo. Another advantage of anti-idiotypic MoAb
is its ability to mark the clonal population of target-specific
lymphocytes. Although tetramers can mark TCR and T-cell clones,
identity of the peptide antigen is required and this technology is
not easily available. Furthermore, anti-idiotypic MoAb can mark
T-cell clones in vivo when radiolabeled, an option not yet possible
with tetramers. Finally, the potential of anti-idiotypic MoAb to
activate the transduced lymphocytes in vivo is appealing,
especially when tumor cells are poorly immunogenic, or when they
are scarcely distributed. Although we used anti-idiotypic MoAb in
our SCID mice experiments, this strategy clearly requires further
optimization after a better understanding of in vivo biology of
these transduced cells become available.
Despite these encouraging results, other structural issues of CIR
technology will have to be considered for future optimization. The
choice of the appropriate spacer (between scFv and signaling
molecule), transmembrane domain and the signaling molecules may be
important (28). That 8H9scFv-modified T-cells proliferate with
anti-idiotype and kill antigen-positive tumor cells argue strongly
that the CD28 trans-membrane domain in this CIR design does not
require a CD8 hinge, permitting effective interaction with soluble
as well as cell-bound antigens. This interaction effects positive
lymphocyte signaling, for both survival and activation, as
previously reported for similar chimeric fusion protein containing
both CD28 and TCR-chains (13). It is possible that the level of
activation could be improved by the addition of a hinge or the
adoption of other trans-membrane domains, as previously suggested
(29). Previous reports have suggested that a human IgG
hinge-CH2-Ch3 spacer can optimize T-cell activity, surface
expression, and target affinity (28, 30). Moreover, using domains
or molecules further downstream in the T-cell activation pathway
could potentially overcome the T-cell defects commonly found in
cancer patients (31). Another variable in T-cell activation is the
affinity of interaction between TCR and MHC peptide complex (32).
Whether a chimeric receptor of low affinity scFv may better mimic
naive TCR interaction needs to be further tested. An optimal
density of CIR for T-cell activation is probably important (33),
since excessive TCR signaling may trigger premature death. In
addition, since most target antigens are not tumor-specific, it may
be useful to standardize the level of expression of CIR such that
an engineered T-cell is optimally activated only by a narrow
threshold of antigen.
The choice of tumor system and antigen target will likely determine
the clinical success of CIR strategy. Primary lymphoid tumors e.g.
B-cell lymphomas have distinct attributes. Because of their innate
tropism, T-cells home to these lymphomas. In addition, these tumors
have unique tumor antigens with homogeneous expression that do not
modulate from the cell surface (e.g. CD20). Furthermore, these
B-cell tumors express costimulatory molecules (30). Most solid
tumors lack these attributes. However, metastatic cancers in lymph
nodes, blood and bone marrow are unique compartments where CIR
technology may be applicable. Depending on the compartment,
targeting of T-cells may require different chemokine receptors or
adhesion molecules. For example, while L-selectin is required for
homing to lymphoid organs, its role for trafficking to other
metastatic organs such as marrow is less well defined.
In adoptive cell therapies, the precise evaluation of the quantity
and persistence of these cells in vivo, as well as their
distribution and function within tissues is critical (34). In
studies of T-cell therapy, this is of particular importance since
many infused cells will undergo activation-induced death in vivo
(35), or immune elimination of gene-modified cells may occur,
especially following repeated injections (36). The development of
sensitive, accurate and reproducible methods to quantify
gene-marked cells in peripheral blood and tissues are essential for
defining the long-term fate of adoptively-transferred cells. While
PCR and quantitative RT-PCR methods are ideal for studying tissues
extracts, anti-idiotypic MoAb will provide useful tools to
enumerate individual scFv-positive cells in blood, marrow and
tumor. In addition, noninvasive imaging methods using radiolabeled
anti-idiotypic MoAb may also be possible. Similar to the marker
gene HSV-tk that allows cells to be tracked and quantified by the
substrate .sup.131I-FIAU or .sup.124I-FIAU, anti-idiotypic MoAb
labeled with either .sup.131I or .sup.124I can also take advantage
of instrumentation and software developed for SPECT and
PET/micro-PET imaging, respectively. These tools can provide
unprecedented precision and dynamic information on cell traffic in
patient trials.
REFERENCES
1. Daniel, P. T., Kroidl, A., Cayeux, S., Bargou, R., Blankenstein,
T., and Dorken, B. Costimulatory signals through B7.1/CD28 prevent
T cell apoptosis during target cell lysis. Immunol, 159: 3808-3815,
1997. 2. Eshhar, Z., Waks, T., Gross, G., and Schindler, D. G.
Specific activation and targeting of cytotoxic lymphocytes through
chimeric single chains consisting of antibody-binding domains and
the gamma or zeta subunits of the immunoglobulin and T-cell
receptors. Proceedings of the National Academy of Sciences of the
United States of America, 90: 720-724, 1993. 3. Stancovski, I.,
Schindler, D. G., Waks, T., Yarden, Y., Sela, M., and Eshhar, Z.
Targeting of T lymphocytes to Neu/HERe-expressing cells using
chimeric single chain Fv receptors. J Immunol, 151: 6577-6582,
1993. 4. Moritz, D., Wels, W., Mattern, J., and Groner, B.
Cytotoxic T lymphocytes with a grafted recognition specificity for
ERBB2-expressing tumor cells. Proc. Natl Acad Sci, USA, 91:
4318-4322, 1994. 5. Wels, W., Moritz, D., Schmidt, M., Jeschke, M.,
Hynes, N. E., and Groner, B. Biotechnological and gene therapeutic
strategies in cancer treatment. Gene, 159: 73-80, 1995. 6. Hwu, P.,
Shafer, G. E., Treisman, J., Schindler, D. G., Gross, G., Cowherd,
R., Rosenberg, S. A., and Eshhar, Z. Lysis of ovarian cancer cells
by human lymphocytes redirected with a chimeric gene composed of an
antibody variable region and the Fc-receptor gamma-chain. J. Exp.
Med., 178: 361-369, 1993. 7. Eshhar, Z., Waks, T., Bendavid, A.,
and Schindler, D. G. Functional expression of chimeric receptor
genes in human T cells. J Immunol Methods, 248: 67-76, 2001. 8.
Rosenberg, S. A. Cell transfer therapy: clinical applications. In:
V. T. J. DeVita, S. Hellman, and S. A. Rosenberg (eds.), Biologic
therapy of cancer, second edition, pp. 487-506. Philadelphia: J.B.
Lippincott Company, 1995. 9. Yang, A.-G. and Chen, S.-Y. A new
class of antigen-specific killer cells. Nat Biotechnol, 15: 46-51,
1997. 10. Culver, K. W., Ram, Z., Wallbridge, S., Ishii, H.,
Oldfield, E. H., and Blaese, R. M. In vivo gene transfer with
retroviral vector-producer cells for treatment of experimental
brain tumors. Science, 256: 1550, 1992. 11. Wei, M. X., Tamiya, T.,
and Chase, M. Experimental tumor therapy in mice using the
cyclophosphamide-activating cytochrome P450 2B1 gene. Hum Gene
Ther, 5: 969, 1994. 12. Weijtens, M. E., Willemsen, R. A., Valerio,
D., Stam, K., and Bolhuis, R. L. Single chain Ig/gamma
gene-redirected human T lymphocytes produce cytokines, specifically
lyse tumor cells, and recycle lytic capacity. J Immunol, 157:
836-843, 1996. 13. Finney, H. M., Lawson, A. D. G., Bebbington, C.
R., and Weir, N. C. Chimeric receptors providing both primary and
costimulatory signaling in T cells from a single gene product. J
Immunol, 161: 2791-2797, 1998. 14. Thanavala, Y. M., Brown, S. E.,
Howard, C. R., Roitt, I. M., and Steward, M. W. A surrogate
hepatitis B virus antigenic epitope represented by a synthetic
peptide and an internal image antiidiotype antibody. Journal of
Experimental Medicine, 164: 227-236, 1986. 15. Wagner, U.,
Schlebusch, H., Kohler, S., Schmolling, J., Grunn, U., and Krebs,
D. Immunological responses to the tumor-associated antigen CA125 in
patients with advanced ovarian cancer induced by the murine
monoclonal anti-idiotype vaccine ACA125. Hybridoma, 16: 33-40,
1997. 16. Modak, S., Kramer, K., Humayun, G., Guo, H. F., and
Cheung, N. K. V. Monoclonal antibody 8H9 targets a novel cell
surface antigen expressed by a wide spectrum of human solid tumors.
Cancer Research, 61: 4048-4054, 2001. 17. Cheung, N. K., Saarinen,
U., Neely, J., Landmeier, B., Donovan, D., and Coccia, P.
Monoclonal antibodies to a glycolipid antigen on human
neuroblastoma cells. Cancer Research, 45: 2642-2649, 1985. 18.
Cheung, N. K., Canete, A., Cheung, I. Y., Ye, J. N., and Liu, C.
Disialoganglioside GD2 anti-idiotypic monoclonal antibodies.
International Journal of Cancer, 54: 499-505, 1993. 19. Towbin, H.,
Staehelin, T., and Gordon, J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Proceedings of the National Academy of Sciences
of the United States of America, 76: 4350-4354, 1979. 20. Krause,
A., Guo, H. F., Tan, C., Cheung, N. K. V., and Sadelain, M.
Antigen-dependent CD-28 signaling enhances survival and
proliferation in genetically modified activated human primary T
lymphocytes. J. Exp. Med., 188: 619-626, 1998. 21. Koehne, G.,
Gallardo, H. F., Sadelain, M., and O'Reilly, R. J. Rapid selection
of antigen-specific T lymphocytes by retroviral transduction.
Blood, 96: 109-117, 2000. 22. Bunnell, B. A., Muul, L. M., Donahue,
R. E., Blaese, R. M., and Morgan, R. A. High-efficiency
retroviral-mediated gene transfer into human nonhuman primate
peripheral blood lymphocytes. Proceeds of the National Academy of
Science, USA, 92: 7739-7743, 1995. 23. Miller, A. D., Garcia, J.
V., von Suhr, N., Lynch, C. M., Wilson, C., and Eiden, M. V.
Construction and properties of retrovirus packaging cells based on
gibbon ape leukemia virus. J Virol, 1991: 2220-2224, 1991. 24. Lam,
J. S., Reeves, M. E., Cowherd, R., Rosenberg, S. A., and Hwu, P.
Improved gene transfer into human lymphocytes using retroviruses
with gibbon ape leukemia virus envelope. Hum Gene Ther, 7:
1415-1422, 1996. 25. Bonini, C., Ferrari, G., Verzeletti, S.,
Servida, P., Zappone, E., Ruggieri, L., Ponzoni, M., Rossini, S.,
Mavilio, F., Traversari, C., and Bordignon, C. HSV-TK gene transfer
into donor lymphocytes for control of allogeneic
graft-versus-leukemia. Science, 276: 1719-1723, 1997. 26. Pollok,
K. E., Hanenberg, H., Noblitt, T. W., Schroeder, W. L., Kato, I.,
Emanuel, D., and Williams, D. A. High-efficiency gene transfer into
normal and adenosine deaminase-deficient T lymphocytes is mediated
by transduction on recombinant fibronectin fragments. J Virol, 72:
4882-4892, 1998. 27. Galea-Lauri, J., Darling, D., Gan, S.-U.,
Krivochtchapov, L., Kuiper, M., Gaken, J., Souberbielle, B., and
Farzaneh, F. Expression of a variant of CD28 on a subpopulation of
human NK cells: implications for B7-mediated stimulation of NK
cells. J Immunol, 163: 62-70, 1999. 28. Patel, S. D., Moskalenko,
M., Smith, D., Maske, B., Finer, M. H., and McArther, J. G. Impact
of chimeric immune receptor extracellular protein domains on T cell
function. Gene Therapy, 6: 412-419, 1999. 29. Fitzer-Attas, C. J.,
Schindler, D. G., Waks, T., and Eshhar, Z. Harnessing Syk family
tyrosine kinases as signaling domains for chimeric single chain of
the variable domain receptors: optional design for T cell
activation. J Immunol, 160: 145-154, 1998. 30. Jensen, M., Tan, G.,
Forman, S., Wu, A. M., and Raubitschek, A. CD20 is a molecular
target for scFvFc: receptor redirected T cells: implications for
cellular immunotherapy of CD20+ malignancy. Biol Blood Marrow
Transplant, 4: 75-83, 1998. 31. Eshhar, Z. and Fitzer-Attas, C. J.
Tyrosine kinase chimeras for antigen-selective T-body therapy. Adv
Drug Deliv Rev, 31: 171-182, 1998. 32. Valitutti, S, and
Lanzavecchia, A. Serial triggering of TCRs: a basis for the
sensitivity and specificity of antigen recognition. Immunology
Today, 18: 299-304, 1997. 33. Varez-Vallina, L. and Russell, S. J.
Efficient discrimination between different densities of target
antigen by tetracycline-regulatable T bodies. Hum Gene Ther, 10:
559-563, 1999. 34. Yee, C., Riddell, S. R., and Greenberg, P. D. In
vivo tracking of tumor-specific T cells. Curr Opin Immunol, 13:
141-146, 2001. 35. Xiaoning, R. T., Ogg, G. S., Hansasuta, P.,
Dong, T., Rostron, T., Luzzi, G., Conlon, C. P., Screaton, G. R.,
McMichael, A. J., and Rowland-Jones, S. Rapid death of adoptively
transferred T cells in acquired immunodeficiency syndrome. Blood,
93: 1506-1510, 1999. 36. Riddell, S. R., Elliott, M., Lewinsohn, D.
A., Gilbert, M. J., Wilson, L., Manley, S. A., Lupton, S. D.,
Overell, R. W., Reynolds, T. C., Corey, L., and Greenberg, P. D.
T-cell mediated rejection of gene-modified HIV-specific cytotoxic T
lymphocytes in HIV-infected patients. Nat Med, 2: 216-223,
1996.
Seventh Series of Experiments
Radioimmunotargeting to Human Rhabdomysarcoma (RMS) Using
Monoclonal Antibody (MoAb) 8H9
Metastatic rhabdomyosarcoma is a chemotherapy-responsive tumor.
However, cure is elusive because of the failure to eradicate
minimal residual disease (MRD). MoAb may have potential for
selective targeting of therapy to MRD. Few MoAb of clinical utility
have been described for RMS. We previously reported the broad tumor
reactivity of a murine MoAb 8H9 with low/no staining of normal
human tissues. The target antigen was typically expressed in a
homogeneous fashion among neuroectodermal (neuroblastoma, Ewing's
sarcoma, PNET, brain tumors), mesenchymal (RMS, osteosarcoma, DSRT,
STS) and select epithelial tumors. Of 25 RMS tumors, 24 stained
positive. Radioimmunolocalization of subcutaneous RMS xenografts in
SCID mice was studied using radiolabeled 8H9. Following iv
injection of 120 uCi of .sup.125I-8H9, selective tumor uptake was
evident at 4 to 172 hrs after injection, with a blood T1/2 of 0.8 h
and T1/2 of 26 h. Mean tumor/tissue ratios were optimal at 172 h
(for lung 4, kidney 7, liver 9, spleen 10, femur 16, muscle 21,
brain 45). Average tumor/blood ratio were 0.7, 1.4 and 1.6, and
tumor uptake was 9.5.+-.3.4, 13.3.+-.1.5, and 5.3.+-.0.9% injected
dose per gm at 24, 48 and 172 h, respectively. The selective
targeting of 8H9 to RMS xenografts suggests its potential for
radioimmunodetection and MoAb-based targeted therapy of MRD in
RMS.
Radioimmunotargeting of Human Rhabdomyosarcoma Using Monoclonal
Antibody 8H9
Abstract
Purpose: Although metastatic rhabdomyosarcoma (RMS) is chemotherapy
and radiotherapy-responsive, few patients are cured. 8H9, a murine
IgG1 monoclonal antibody (MoAb), recognizes a unique cell surface
antigen that has restricted expression on normal tissues but is
broadly distributed on neuroectodermal, epithelial and mesenchymal
tumors including RMS. In this report we test its immunotargeting
potential in mice with subcutaneous human RMS.
Experimental design: Athymic nude mice with established RMS
xenografts were injected intravenously with .sup.125I-8H9 or
.sup.125I-control MoAb. .sup.125I-8H9 immunoreactivity was tested
on solid-phase anti-8H9-idiotypic rat MoAb 2E9. Mice were imaged
using a gamma camera and biodistribution of radiolabeled antibodies
determined. The anti-tumor effect was studied following intravenous
(IV) administration of 18.5 MBq 1311-8H9.
Results: Following IV injection of 4.44 MBq of .sup.125I-8H9,
selective tumor uptake was evident 4 to 172 h after injection.
Average tumor uptake was 11.5.+-.3.9, 15.1.+-.3.7, and 5.4.+-.1.2%
injected dose per gm at 24, 48 and 172 h, respectively. Mean
tumor/tissue ratios were optimal at 172 h (for lung, 4, kidney 6,
liver 7, spleen 11, femur 14, muscle 18, brain 48). Tumor/tissue
ratios were improved when a lower dose (0.74 MBq) of .sup.125I-8H9
was injected. No hematological or histological abnormalities were
observed. Mice injected with .sup.125I-negative control did not
demonstrate specific tumor uptake. In contrast to .sup.131I-control
treated mice, which showed unabated tumor progression, mice treated
with 18.5 MBq of .sup.131I-8H9 showed tumor suppression of
>50%.
Conclusions: Radiolabeled 8H9 effectively targeted RMS xenografts
and may have a potential clinical role in immunodetection and
immunotherapy.
Introduction
Metastatic rhabdomyosarcoma (RMS) is associated with a dismal
prognosis with reported cure rates of no greater than 25% despite
demonstrated chemosensitivity and radiosensitivity (1,2,3).
Myeloablative chemotherapy with autologous stem cell rescue has
failed to impact survival (4,5). The failure to eradicate minimal
residual disease (MRD) leads to local and distant relapses for both
alveolar and embryonal RMS. Alternative strategies to target MRD
are therefore warranted. Monoclonal antibodies (MoAbs) have
recently been reported to be of clinical benefit in the treatment
of solid tumors. In children with high-risk neuroblastoma (NB), the
addition of the anti-ganglioside G.sub.D2 antibody 3F8 to a
multimodality approach has significantly improved prognosis (6)
without increasing long-term toxicity (7). Radiolabeled antibodies
can selectively deliver radiation to human tumors. Demonstration of
specific binding to NB xenografts by .sup.131I-3F8 was initially
demonstrated in xenograft models (8). Indeed, .sup.131I-3F8
completely ablated NB xenografts in athymic nude mice with
reversible toxicity (9). Based on the pharmacokinetics and
dosimetry calculations to tumors and normal tissues
radioimmunodetection and radioimmunotherapy, clinical protocols
utilizing .sup.131I-3F8 were initiated in patients with NB.
Subsequently, effective and specific targeting of NB in humans was
demonstrated (10,11), and later utilized both for detection and
therapy.
The adoption of a similar strategy to RMS has been limited by the
paucity of antigens that can be targeted by MoAbs. Most antigens
expressed on RMS either have a nuclear or cytoplasmic localization
which makes them inaccessible to MoAbs, or are coexpressed on
normal tissues thus limiting their clinical utility (Table 1). The
PAX-FKHR fusion transcript is specific for alveolar RMS. It has
been used in the detection of micrometastases in alveolar RMS by
RT-PCR (12, 13) and as a tumor antigen for the generation of
cytotoxic T-cells (14). However, its nuclear localization shields
the intact protein from antibody-based targeting approaches.
Furthermore, for the more frequent embryonal variant, such specific
markers are not yet available. We recently described a novel tumor
antigen with an apparent molecular weight of 58 kD (15) recognized
by the MoAb 8H9. This glycoprotein is expressed on cell surface of
a broad spectrum of solid tumors in childhood and adults, including
both alveolar and embryonal RMS and has restricted distribution on
normal tissues. We now report the in vivo targeting of .sup.125I
and .sup.131I labeled 8H9 in human RMS xenografts.
TABLE-US-00026 TABLE 1 Previously reported antigens on
rhabdomyosarcoma Antigen Localization Crossreactivity Desmin (22)
cytoplasm Skeletal, Cardiac and Smooth Muscle Cytokeratin (23)
cytoplasm Epithelial cells EMA (23) cytoplasm Epithelial cells
Vimentin (23) cytoplasm All mesenchymal tissues NSE (23) cytoplasm
Brain and neural tissue MYOD1 (17) nucleus Restricted to RMS Ag
BW575 (18) cell membrane Neural cells Myosin (19) cell membrane
Muscle cells 5.1 H11 (25) cytoplasm Neural cells IGFI receptor (21)
cell membrane Normal cells Fetal acetylcholine receptor cell
membrane Extraocular muscles, (20) thymus, denervated skeletal
muscle
TABLE-US-00027 TABLE 2 % injected dose/gram of .sup.125I-8H9
distributed in HTB82 xenografts and normal tissues 24, 48 and 172
hours after injection 172 h 24 h (n = 9 mice) 48 h (n = 9 mice) (n
= 8 mice) Mean .+-. SD Mean .+-. SD Mean .+-. SD Adrenal 1.4 .+-.
1.6 1.4 .+-. 0.5 0.4 .+-. 0.3 Bladder 2.6 .+-. 1.2 2.9 .+-. 0.8 0.9
.+-. 0.6 Blood 14.1 .+-. 3.0 10.7 .+-. 2.1 3.2 .+-. 0.9 Brain 0.3
.+-. 0.1 0.3 .+-. 0.1 0.1 .+-. 0.0 Femur 1.4 .+-. 0.5 1.1 .+-. 0.5
0.4 .+-. 0.1 Heart 4.3 .+-. 1.9 2.9 .+-. 0.5 0.9 .+-. 0.2 Kidney
3.9 .+-. 1.6 3.0 .+-. 0.7 0.8 .+-. 0.3 Large Intestine 1.7 .+-. 0.6
1.2 .+-. 0.3 0.2 .+-. 0.1 Liver 4.0 .+-. 1.7 2.2 .+-. 0.3 0.7 .+-.
0.3 Lung 5.7 .+-. 3.5 5.3 .+-. 1.1 1.4 .+-. 0.5 Muscle 1.2 .+-. 0.6
1.1 .+-. 0.4 0.3 .+-. 0.1 Skin 2.3 .+-. 1.6 2.5 .+-. 1.5 0.6 .+-.
0.4 Small Intestine 1.5 .+-. 0.4 1.1 .+-. 0.2 0.3 .+-. 0.1 Spine
2.1 .+-. 0.8 1.7 .+-. 0.7 0.5 .+-. 0.2 Spleen 5.8 .+-. 2.4 3.3 .+-.
0.8 0.5 .+-. 0.2 Stomach 2.4 .+-. 2.1 1.6 .+-. 0.7 0.5 .+-. 0.4
Tumor 11.5 .+-. 3.9 15.1 .+-. 3.7 5.4 .+-. 1.2
TABLE-US-00028 TABLE 3 Tumor:non-tumor ratios in mice injected with
0.74MBq compared to 4.44MBq of .sup.125I-8H9 172 h post injection
(5 mice per group) 0.74MBq 4.44MBq Mean .+-. SD Mean .+-. SD
Adrenal 26.3 .+-. 20.4 12.5 .+-. 3.6 Bladder 35.0 .+-. 31.4 7.9
.+-. 1.5 Blood 2.6 .+-. 1.7 1.7 .+-. 0.3 Brain 150.9 .+-. 36.1 51.9
.+-. 20.1 Femur 26.7 .+-. 20.6 13.7 .+-. 2.0 Heart 11.5 .+-. 8.5
5.7 .+-. 1.0 Kidney 8.4 .+-. 3.5 6.5 .+-. 1.4 Large Intestine 32.3
.+-. 18.6 23.0 .+-. 5.0 Liver 13.0 .+-. 6.7 7.0 .+-. 0.9 Lung 7.7
.+-. 6.0 4.1 .+-. 0.6 Muscle 33.0 .+-. 22.3 18.9 .+-. 4.4 Skin 13.0
.+-. 8.9 7.2 .+-. 3.1 Small Intestine 29.4 .+-. 17.0 20.8 .+-. 6.4
Spine 20.4 .+-. 11.1 10.3 .+-. 3.7 Spleen 16.4 .+-. 11.3 11.9 .+-.
2.0 Stomach 23.4 .+-. 15.9 14.5 .+-. 4.3 Tumor 1.0 .+-. 0 1.0 .+-.
0
TABLE-US-00029 TABLE 4 .sup.125I-8H9 and .sup.125I-HTB82 xenografts
(values represent dose/gram). .sup.125I-8H9 .sup.125I-2C9 Mean .+-.
SD Mean .+-. SD Adrenal 0.5 .+-. 0.2 0.7 .+-. 0.4 Bladder 1.5 .+-.
0.8 1.5 .+-. 0.4 Blood 4.6 .+-. 0.7 8.4 .+-. 1.4 Brain 0.1 .+-. 0.1
0.2 .+-. 0.1 Femur 0.6 .+-. 0.1 0.9 .+-. 0.2 Heart 1.0 .+-. 0.2 1.7
.+-. 0.4 Kidney 1.2 .+-. 0.3 1.4 .+-. 0.4 Large intestine 0.4 .+-.
0.3 0.5 .+-. 0.1 Liver 0.9 .+-. 0.1 1.4 .+-. 0.1 Lung 2.9 .+-. 0.7
5.3 .+-. 1.5 Muscle 0.4 .+-. 0.1 0.5 .+-. 0.1 Skin 0.8 .+-. 0.1 1.0
.+-. 0.3 Skin 0.8 .+-. 0.1 1.0 .+-. 0.3 Small intestine 0.4 .+-.
0.1 0.6 .+-. 0.1 Spine 0.6 .+-. 0.1 1.3 .+-. 0.5 Spleen 1.3 .+-.
0.6 2.2 .+-. 0.5 Stomach 0.5 .+-. 0.2 1.1 .+-. 0.2 Stomach contents
0.3 .+-. 0.1 0.3 .+-. 0.2 Tumor 7.2 .+-. 0.9 2.5 .+-. 0.9
Biodistribution of 2C9 in mice with 120 h after injection %
injected
TABLE-US-00030 TABLE 5 Mean hematological and liver function
parameters in mice (5 per group) injected with .sup.131I-8H9
Reported Day 15 Day 30 normal values CBC Hb (g/dl) 11.2 .+-. 0.3
13.1 .+-. 3.2 11.0-14.0 WBC (10.sup.3) 4.43 .+-. 0.7 6.2 .+-. 2.7
2.8-9.2 Platelets (10.sup.3) 1309 .+-. 371 1300 .+-. 798 1523 .+-.
218 Segmented (%) 46.8 .+-. 9.9 42.5 .+-. 11.4 42-45.5 Lymphocytes
(%) 49.6 .+-. 11.6 51.2 .+-. 16.2 54.5-58 Liver function tests
(pooled serum) Alk. Phosphatase (IU/L) 96 174 66-258 ALT (IU/L) 36
33 62-121 AST (IU/L) 169 93 87-318 GGT (IU/L) 0 0 Albumin (g/dl)
3.1 4.8 2.5-4.8 Total protein (g/dl) 5.5 5.1 3.5-7.2 Total
bilirubin (mg/dl) 0.1 0.3 0.1-0.9
Materials and Methods Monoclonal Antibodies
MoAb 8H9 The murine MoAb 8H9 was produced by hyperimmunizing BALB/c
mice with human neuroblastoma as previously described. (15).
MoAb 2C9 Using similar methods, mice were immunized with human
breast cancer and the hybridoma demonstrating specificity against
cytokeratin 8 was isolated.
Anti-idiotypic MoAbs Rat anti-8H9-idiotype MoAbs were produced by
immunizing LOU/CN rats with purified 8H9. Following in vitro
hybridization with the myelomas SP2/0 or 8653, three IgG.sub.2a
clones (2E9, 1E12 and 1F11) were selected for their high binding
and specificity by ELISA. When tested against a panel of 23 other
myelomas, no crossreactivity was found. The anti-idiotypic
hybridomas were cloned and the antibody 2E9 chosen for scaled up
production using high-density MiniPERM bioreactor (Unisyn
technologies, Hopkinton, Mass.). Anti-idiotypic antibodies were
further purified by protein G affinity (Hitrap G, Pharmacia,
Piscataway, N.J.) chromatography and filtered through a 0.2 nm
Millipore filter (Millipore Inc., Bedford, Mass.).
Cell Lines
RMS cell line HTB82 and small cell lung cancer cell line HTB119
(8H9 negative control) were purchased from American Type Culture
Collection, Bethesda, Md. Cell lines were grown in RPMI (Gibco BRL,
Gaithersburg, Md.) supplemented with 10% newborn calf serum
(Hyclone, Logan, Pa.), 2 mM glutamine, 100 U/ml penicillin and 100
ug/ml streptomycin (Gibco-BRL, Gaithersburg, Md.). Cells were
cultured in a 37.degree. C. incubator and harvested using 2 mM
EDTA.
Iodination
MoAb 8H9 was allowed to react for 5 min with .sup.1251 or .sup.1311
(NEN Life Sciences, Boston, Mass.) and chloramine T (1 mg/ml in
0.3M Phosphate buffer, pH 7.2) at room temperature. The reaction
was stopped by adding sodium metabisulfite (1 mg/ml in 0.3M
Phosphate buffer, pH 7.2) for 2 min Radiolabeled MoAb was separated
from free iodine using A1GX8 resin column (BioRad, Richmond,
Calif.) saturated with 1% HSA (New York Blood Center Inc., Melville
Biologics Division, New York, N.Y.) in PBS, pH 7.4. Peak
radioactive fractions were pooled and the radioactivity (MBq/ml)
was measured using a radioisotope calibrator (Squibb, Princeton,
N.J.). Iodine incorporation and specific activities were
calculated. Trichloroacetic acid (TCA) (Fisher Scientific,
Pittsburgh, Pa.) precipitation was used to assess the percentage of
protein bound .sup.125I or .sup.131I. Thin layer chromatography was
performed by running 1 .mu.l of .sup.125I-8H9 on a silica gel on
glass TLC plate (Sigma Chemical, St. Louis, Mo.) and scanning it
with System 200 Imaging Scanner (Bioscan, Washington, D.C.).
In Vitro Immunoreactivity of Iodinated 8H9
Immunoreactivity of labeled antibody was determined by a specific
microtiter solid phase radioimmunoassay developed using the
anti-8H9-idiotypic antibody 2E9 as the antigen. Briefly, microtiter
plates were precoated with diminishing concentrations of 2E9.
Appropriate dilutions of .sup.125I-8H9 were added in duplicate.
Binding was maximized by serial incubations at 4.degree. C. in 3
separate antigen plates for periods of 1 h, 4 h and overnight
respectively. The percent of bound activity was summed for each
dilution to obtain the maximum percent binding. Similar assay was
carried out to assess immunoreactivity of .sup.131I-8H9.
Immunoreactivity was also measured by specific binding to cell
pellets. HTB82 cells were suspended in Eppendorff tubes at
concentrations of 10.sup.8, 10.sup.7 and 10.sup.6/ml in 100 .mu.l
medium. 100 .mu.l of appropriate dilution of .sup.125I-8H9 was
added and allowed to react at 37.degree. C. for 60 mins. Tubes were
subsequently centrifuged at 1400 rpm.times.10 mins Radioactivity in
100 .mu.l of supernatant was counted using Minaxi gamma counter
(Packard BioScience, Downer's Grove, Ill.) and compared with total
counts in a control sample consisting of medium without cells.
Percent binding was calculated as (Experimental cpm/control
cpm).times.100%. The 8H9-negative cell line HTB 119 was used as
control.
Animal Studies
Biodistribution and Pharmacokinetics
All animal experiments were carried out under an IACUC approved
protocol and institutional guidelines for the proper and humane use
of animals in research were followed. Athymic nude mice (Ncr nu/nu)
were purchased from NCI, Frederick Md. They were xenografted
subcutaneously with HTB82 cell line (2.times.10.sup.6 cells/mouse)
suspended in 100 ul of Matrigel (Becton-Dickinson BioSciences,
Bedford, Mass.) on the right flank. After 3-4 weeks, mice bearing
tumors of 1 to 1.5 cm in longest dimension were selected. Mice were
injected intravenously (retrorbital plexus) with 0.74 MBq or 4.44
MBq of .sup.125I-8H9, or with 4.44 MBq .sup.125I-2C9. They were
anesthetized with ketamine (Fort Dodge Animal Health, Fort Dodge,
Iowa) intraperitoneally and imaged at various time intervals with a
gamma camera (ADAC, Milpitas, Calif.) equipped with a
high-resolution general-purpose collimator for .sup.131I and
thyroid X-ray grids for .sup.125I. Serial blood samples were
collected at 5 min, 1, 2, 4, 8, 18, 24, 48, 72, 120, 144 and 172 h
to determine blood clearance of .sup.125I-8H9. Groups of mice
injected with .sup.125I-8H9 were sacrificed at 24 h, 48 h, 120 h or
172 h immediately after imaging. Mice injected with .sup.125I-2C9
were imaged either at 120 h (and then sacrificed) or at 172 h.
Samples of blood (cardiac sampling), heart, lung, liver, kidney,
spleen, stomach, adrenal, small bowel, large bowel, spine, femur,
muscle, skin, brain and tumor were weighed and radioactivity
measured with a Minaxi-gamma counter. Results were expressed as
percent injected dose per gram and biodistribution determined.
Toxicity
Athymic nude mice without xenografts were each injected with 4.44
MBq of .sup.131I-8H9. Groups of mice were euthanized at 15 and 30
days. Complete blood counts were carried out in each mouse via
terminal bleed and liver function tests were performed on pooled
sera. Complete necropsies including gross and histological
examinations were carried out to evaluate possible toxicity of
.sup.131I-8H9.
Evaluation of Anti-Tumor Activity
RMS xenografts were established as described above. Their maximal
perpendicular axes were measured using calipers in control and
tumor groups. After 3 weeks, mice bearing tumors of approximately
0.7 cm.sup.3 (tumor volume was calculated using the formula
V=4.pi.r.sup.3/3 where r=mean radius) were selected and injected
with 18.5 MBq of .sup.131I-8H9 or .sup.131I-3F8 (3F8 was used as a
negative control antibody). Average serial tumor volumes and body
weights were monitored in the two groups and compared over time.
Mice were euthanized as per guidelines published in NIH Publication
No. 85-23 ('Principles of Laboratory Animal Care'). Data are
expressed as % increase or decrease in tumor volume when compared
to initial measurement on day 0 of treatment.
Results
Immunoreactivity
Protein bound .sup.125I and .sup.131I as assessed by TCA
precipitation averaged 96.+-.4.2% and 98.+-.2.2%, respectively for
8H9, and >95% for control antibodies 2C9 and 3F8. TLC
demonstrated free iodine peak of 1%, 99% being protein bound.
Average maximum immunoreactivity as measured by solid-phase RIA
using the anti-8H9-idiotype 2E9 as antigen was 67.+-.26% for 8H9
and 11% for 2C9. Maximum immunoreactivity measured by cell pellet
binding assay was 83% for 8H9, maximum binding to the negative
control cell line HTB119 being 9%. 2C9 demonstrated maximum binding
of 6% on the HTB82 cell pellet.
Imaging
Animals tolerated intravenous injection without apparent ill
effects. Tumor localization could be detected in animals imaged
with .sup.125I-8H9 as early as 4 hours after injection. At 24 h,
tumor localization was obvious along with some blood pool, liver
and spleen uptake. At 48 h, blood pooling had significantly
diminished and almost disappeared at 172 h. In contrast, mice
injected with the control IgG1 .sup.125I-2C9 demonstrated no
specific uptake in RMS xenografts (FIG. 28).
Blood Kinetics
Average blood clearance in groups of 5 mice with and without RMS
xenografts injected with .sup.125I-8H9 is depicted in FIG. 29.
Blood activity of .sup.125I-8H9 at 24 h was 14.3% and 17.3%
injected dose per gm (% ID/g) respectively and dropped off to 3.3%
and 5.3% ID/g, respectively at 172 h. .beta. half-life of
.sup.125I-8H9 was 70.9 h.
Biodistribution
Table 2 lists the biodistribution of 4.44 MBq .sup.125I-8H9 in
three groups of mice with RMS xenografts studied at 24, 48 and 172
h, respectively. Blood-pooling effect was observed at 24 h, which
had diminished at 48 h and had almost completely subsided at 172 h
after injection. There was no significant activity in normal organs
apart from blood at 172 h. Average tumor uptake was 11.5.+-.3.9,
15.1.+-.3.7, and 5.4.+-.1.2% injected dose per gm at 24, 48 and 172
h, respectively. Blood to tumor ratio was 1.24, 0.71 and 0.59 at
24, 48 and 172 h respectively. Mean tumor/tissue ratios (FIG. 30)
increased from 24 to 48 h and were optimal at 172 h (for lung, 4,
kidney 7, liver 8, spleen 11, femur 15, muscle 20, brain 47). In
mice injected with 0.74 MBq .sup.125I-8H9, there was a further
increase in tumor:tissue ratios particularly marked at 172 h post
injection (for lung, 6, kidney 8, liver 12, spleen 14, femur 21,
muscle 28, brain 56) (Table 3). Table 4 summarizes the
biodistribution of .sup.125I-8H9 compared to .sup.125I-2C9 at 120 h
post injection. Average tumor uptake was 7.3.+-.0.9% injected dose
per gram for .sup.125I-8H9 as compared to 2.5.+-.0.9% for
.sup.125I-2C9. Tumor to tissue ratios (FIG. 31) were <1 for
almost all tissues for .sup.125I-2C9, as compared to 2.6-56.0 for
.sup.125I-8H9.
Anti-Tumor Activity
Mice injected with 18.5 MBq .sup.131I-8H9 showed a significant
suppression in tumor volume (FIG. 32). Average tumor volume had
diminished to <50% of initial volume 21 days after injection.
None of the tumors showed any evidence of regrowth. In contrast, in
the control group, mice injected with 18.5 MBq of .sup.131I-3F8, an
anti-GD2 MoAb that does not react with HTB82, there was progressive
and rapid tumor growth.
Toxicity
No significant weight loss was noted in mice injected with 4.44 MBq
of .sup.131I-8H9, 15 and 30 days post injection (data not shown).
Complete blood count and liver function studies did not reveal any
abnormalities (Table 5). Complete necropsy evaluations did not
reveal any gross or histological lesions (data not shown). In the
groups of mice treated with .sup.131I labeled MoAbs, there was no
significant weight loss 21 days after the initial dose for both the
3F8 and 8H9 groups (+11.7.+-.8.8% for the 3F8 group and -2.+-.1.8%
for the 8H9 group). The increase in weight in the control group
could be attributed to increasing tumor mass.
Discussion
Few tumor specific antigens that can be targeted by MoAbs have been
described for RMS. (Table 1) Myogenin, a myogenic regulatory
protein specific for rhabdomyoblasts is nuclear in localization
(16) and therefore not amenable for targeting by MoAbs. Similarly,
the MyoD family of oncofetal proteins is expressed in nuclei (17).
Conversely, the cell membrane-expressed antigens, BW475 (18) and
myosin (19), are also expressed on normal neural and muscle tissue
respectively. The fetal form of the acetylcholine receptor,
.alpha.2.beta..gamma..delta., a possible target for antibody-based
immunotherapy, although not present on most normal muscles tissue,
is expressed in extraocular muscles, thymic myoid cells and in
denervated skeletal muscle. (20.) Blockade of the insulin-like
growth factor I (IGFI) receptor, which has been implicated in an
autocrine pathway in the growth of RMS by murine monoclonals has
been demonstrated to inhibit the growth of established RMS
xenografts in nude mice (21). However, IGF receptors are
ubiquitously expressed in normal tissues.
MoAb 8H9 recognizes a unique cell membrane antigen which is
expressed on a wide range of pediatric and adult solid tumors (15).
Furthermore, this novel antigen has restricted expression on normal
tissues. In particular skeletal muscle and hematopoietic tissues
are negative. Indeed 8H9 has been utilized to purge Ewing's sarcoma
from blood and bone marrow (26). In RMS, the 8H9 antigen is
expressed on both alveolar and embryonal variants. 96% (29/30) RMS
studied expressed the 8H9 antigen. Expression in most cases was
strong and homogeneous. RMS cell lines including the HTB82 cell
line have been shown to express this antigen on cell membranes. It
therefore has the potential to be utilized as a tumor target in
RMS.
RMS is a chemosensitive and radiosensitive tumor, yet in patients
with metastatic disease, MRD often leads to relapse and prevents
cure. Immunotherapy using radiolabeled and unlabeled 8H9 may
provide a valuable adjunct in the eradication of MRD. A similar
approach has led to successful cures being achieved in high-risk
neuroblastoma (6). In this study we evaluated the in vivo targeting
of RMS by radiolabeled 8H9. We have demonstrated that radiolabeled
8H9 can be effectively used in the radioimmunodetection and
radioimmunotherapy of RMS xenografts in mice. Our results showed
that .sup.125I or .sup.131I labeled 8H9 retained immunoreactivity
after radiolabeling. A relatively high specific activity of >370
MBq/mg of .sup.125I was obtained without loss of immunoreactivity.
Hence, 8H9 has the potential to be labeled with relatively large
doses of iodine radioisotopes for radioimmunotherapy
approaches.
Our imaging results show that 8H9 can specifically and selectively
bind to human RMS xenografted in nude mice. Uptake in xenografts
could be detected as early as 4 h after injection. Excellent
selectivity for tumor over normal tissue was demonstrated. There
was no focal uptake in any normal organs including
reticuloendothelial tissues. This is in keeping with the specific
distribution of the antigen recognized by 8H9 as demonstrated by
immunohistochemistry in human tissues and tumors (15). Specificity
of 8H9 binding was demonstrated by comparing the binding of
.sup.125I-8H9 to that of .sup.125I-2C9. 2C9, an IgG1 MoAb specific
for cytokeratin8, an antigen not expressed by the RMS cell line
HTB82, was used as a negative control. As expected, radiolabeled
2C9 remained in the bloodstream and did not show any specific
binding for RMS xenografts with tumor: tissue ratios of 0.1-1. In
comparison, radiation dose to tumor relative to normal tissues for
125I-8H9 ranged from 2.6 to 25.3 fold. Specificity of .sup.125I-8H9
was also demonstrated in vitro by studying the binding of
.sup.125I-8H9 to the 8H9 negative cell line HTB119 in comparison to
the 8H9 positive line HTB82. Maximum binding of .sup.125I-8H9 was
83% in comparison to 9% for HTB119 indicating that .sup.125I-8H9
binding was antigen specific.
Biodistribution studies provided us with preclinical data in
consideration of a possible use for 8H9 in human trials. .beta.
half-life of a single dose of 4.4 MBq of .sup.125I-8H9 was 70.9 h.
There was, in general, an excellent radiation dose differential
between RMS and normal tissues. Optimum tumor to non-tumor ratios
were reached at 172 h after intravenous 8H9 injection. Blood:tumor
ratios were relatively high at 24 h indicative of blood pooling.
Blood pooling diminished 48 h after injection and was further
greatly reduced by 172 h. Probable uptake by cells of the
reticuloendothelial system resulted in relatively high levels for
liver and spleen in the first 24 h. There was increased uptake in
the tumors at 48 h compared to 24 h suggesting further selective
targeting of 8H9 between 24 to 48 h. Persistence of .sup.125I-8H9
in the blood during the first 48 h of administration implies that
there is no appreciable neutralization of antibody by circulating
8H9 antigen. When lower doses of .sup.125I-8H9 for imaging (0.74
MBq compared to 4.44 MBq), tumor: non-tumor ratios were improved,
consistent with reduced blood pooling (Table 4). The persistence of
binding of .sup.125I-8H9 to tumor implies that the 8H9 antigen is
not immunomodulated off the cell after antibody binding. Similar
findings were demonstrated in vitro, where the antigen-antibody
binding on cell surface as detected by immunofluorescence persisted
>60 h (15). This persistence should permit a steady delivery of
radiation to tumor cells by radiolabeled 8H9. At doses of 6.66
MBq/m.sup.2, there were no clinical (body weights), chemical (CBC
and LFTs) or gross or histologic organ toxicities at
necropsies.
In an effort to develop systems to study antigen-antibody reactions
pending the definitive identification of the glycosylated 58 kDa
protein antigen recognized by 8H9, we used anti-8H9-idiotypic MoAbs
to serve as surrogate antigens. These have enabled us to study the
binding of radiolabeled (radioimmunoassay) and unlabeled (ELISA)
8H9 in vitro. Our data indicate that there was good correlation
between the binding of .sup.125I-8H9 to anti-8H9 anti-idiotypes and
to native antigen on cell pellets.
The observed radioimmunotherapeutic effect of .sup.131I-8H9 was
remarkable, with >50% reduction in tumor volume of
well-established RMS xenografts being achieved with a dose of 18.5
MBq of .sup.131I-8H9 without any adverse effects. The antigen
specific nature of this response was confirmed when RMS xenografts
treated with equivalent doses of nonspecific antibody demonstrated
unabated tumor growth. Radiolabeled 8H9 therefore, may have a
possible clinical role in the therapy of RMS.
Given the broad reactivity of MoAb 8H9 with human solid tumors
including sarcomas, neuroblastoma and brain tumors, these studies
provide the proof of principle for exploring antibody-based
targeting strategies directed at this antigen.
REFERENCES
Crist W, Gehan E A, Ragab A H, Dickman P S, Donaldson S S, Fryer C,
Hammond D, Hays D M, Herrmann J and Heyn R. The Third Intergroup
Rhabdomyosarcoma Study J Clin Oncol 13:610-30, 1995 Maurer H M,
Gehan E A, Beltangady M, Crist W, Dickman P S, Donaldson S S, Fryer
C, Hammond D, Hays D M and Herrmann J. The Intergroup
Rhabdomyosarcoma Study-II. Cancer 71:1904-22, 1993 Okamura J, Sutow
W W, and Moon T E. Prognosis in children with metastatic
rhabdomyosarcoma. Med Pediatr Oncol 3:243-51, 1977 Weigel B J,
Breitfeld P P, Hawkins D, Crist W M, and Baker K S. Role of
high-dose chemotherapy with hematopoietic stem cell rescue in the
treatment of metastatic or recurrent rhabdomyosarcoma. J Pediatr
Hematol Oncol 23:272-276, 2001 Ruymann F B and Grovas A C. Progress
in the diagnosis and treatment of rhabdomyosarcoma and related soft
tissue sarcomas. Cancer Invest 18:223-241, 2000 Cheung N K, Kushner
B H, Cheung I Y, Kramer K, Canete A, Gerald W, Bonilla M A, Finn R,
Yeh S J, and Larson S M. Anti G.sub.D2 antibody treatment of
minimal residual stage 4 neuroblastoma diagnosed at more than 1
year of age. J. Clin. Oncol., 16: 3053-60, 1998 Cheung N K, Kushner
B H, Yeh S D J, and Larson S M. 3F8 monoclonal antibody treatment
of patients with stage 4 neuroblastoma: a phase II study. Int. J.
Oncol 12: 1299-306, 1998 Cheung N K, Neely J E, Landmeier B, Nelson
D and Miraldi F. Targeting of ganglioside GD2 monoclonal antibody
to neuroblastoma J Nuc Med 28:1577-83, 1987 Cheung N K, Landmeier
B, Neely J, Nelson A D, Abramowsky C, Ellery S, Adams R B and
Miraldi F. Complete tumor ablation with iodine 131-radiolabeled
disialoganglioside GD2-specific monoclonal antibody against human
neuroblastoma xenografted in nude mice. J Natl Cancer Inst.
77:739-745, 1986 Yeh S D, Larson S M, Burch L, Kushner B H,
LaQuaglia M, Finn R and Cheung N K. Radioimmunodetection of
neuroblastoma with iodine-131-3F8: correlation with biopsy,
iodine-131-metaiodobenzylguanidine and standard diagnostic
modalities. J. Nucl. Med. 32: 769-76, 1991 Kramer K, Cheung N K,
Humm J L, Dantis E, Finn R, Yeh S J, Antunes N L, Dunkel I J,
Souwedaine M and Larson S M. Targeted radioimmunotherapy for
leptomeningeal cancer using (131) I-3F8. Med Pediatr Oncol
35:716-8, 2000 Thomson B, Hawkins D, Felgenhauer J, and Radich J.
RT-PCR evaluation of peripheral blood, bone marrow and peripheral
blood stem cells in children and adolescents undergoing VACIME
chemotherapy for Ewing's sarcoma and alveolar rhabdomyosarcoma.
Bone Marrow Transplant 24:527-33, 1999 Athale U H, Shurtleff S A,
Jenkins J J, Poquette C A, Tan M, Downing J R and Pappo A S. Use of
Reverse Transcriptase Polymerase Chain Reaction for Diagnosis and
Staging of Alveolar Rhabdomyosarcoma, Ewing Sarcoma Family of
Tumors, and Desmoplastic Small Round Cell Tumor. Am J Pediatr
Hematol Oncol 23(2):99-104, 2001 Mackall C, Berzofsky J and Helman
L J. Targeting tumor specific translocations in sarcomas in
pediatric patients for immunotherapy. Clin Orthop. 373:25-31, 2000
Modak S, Kramer K, Gultekin S H, Guo H F and Cheung N K. Monoclonal
antibody 8H9 targets a novel cell surface antigen expressed by a
wide spectrum of human solid tumors. Cancer Res. 61:4048-54, 2001.
Kumar S, Perlaman, E, Harris C A, Raffeld M and Tsokos M. Myogenin
is a specific marker for rhabdomyosarcoma: an immunohistochemical
study in paraffin embedded tissues. Mod Pathol 13: 988-93, 2000
Wang N. P., Marx J., McNutt M. A., Rutledge J. C., and Gown A. M.,
Expression of myogenic regulatory proteins (myogenin and MyoD1) in
small blue round cell tumors of childhood. Am J Pathol 147:
1799-1810, 1995 Fujisawa T, Xu Z. J., Reynolds C. P Schultz G.,
Bosslet I. V., and. Seeger R. C., A monoclonal antibody with
selective immunoreactivity for neuroblastoma and rhabdomyosarcoma.
(abs) Proc. AACR 30: 345, 1989 Gruchala A, Niezabitowski A,
Wasilewska A, Sikora K, Rys J, Szklarski W, Jaszcz A, Lackowska B
and Herman K. Rhabdomyosarcoma. Morphologic, immunohistochemical,
and DNA study. Gen Diagn Pathol 1142:175-84, 1997. Gattenloehner S,
Vincent A, Leuschner I, Tzartos S, Muller-Hermelink H K, Kirchner T
and Marx A. The fetal form of the acetylcholine receptor
distinguishes rhabdomyosarcomas from other childhood tumors. Am J
Pathol. 152:437-44, 1998 Kalebic T, Tsokos M, Helman L J. In vivo
treatment with antibody against IGF-1 receptor suppresses growth of
human rhabdomyosarcoma and down-regulates p34cdc2. Cancer Res
54:5531-4, 1994 Truong L D, Rangdaeng S, Cagle P, Ro J Y, Hawkins
H, Font R L. The diagnostic utility of desmin A study of 584 cases
and review of the literature Am J Clin Pathol 93:305-14, 1990
Qualman S J, Coffin C M, Newton W A, Hojo H, Triche T J, Parham D
M, and Crist W M. Intergroup Rhabdomyosarcoma Study: update for
pathologists Pediatr Dev Pathol 1:550-61, 1998 Garin-Chesa P.,
Fellinger E. J., Huvos A. G., Beresford H. R., Melamed M. R.,
Triche T. J., and Rettig W. J., Immunohistochemical analysis of
neural cell adhesion molecules. Differential expression in small
round cell tumors of childhood and adolescence. Am. J. Pathol. 139:
275-286, 1991 Strother D R, Parham D M and Houghton P J. Expression
of the 5.1H11 antigen, a fetal muscle surface antigen, in normal
and neoplastic tissue. Arch Pathol Lab Med 114:593-596, 1990 Merino
M E, Navid F, Christensen B L, Toretsky J A, Helman L J, Cheung N K
and Mackall C L. Immunomagnetic purging of ewing's sarcoma from
blood and bone marrow: quantitation by real-time polymerase chain
reaction. J Clin Oncol 19:3649-3659, 2001
SEQUENCE LISTINGS
1
401731DNAMus musculus 1caggtcaaac tgcagcagtc tggggctgaa ctggtaaagc
ctggggcttc agtgaaattg 60tcctgcaagg cttctggcta caccttcaca aactatgata
taaactgggt gaggcagagg 120cctgaacagg gacttgagtg gattggatgg
atttttcctg gagatggtag tactcaatac 180aatgagaagt tcaagggcaa
ggccacactg actacagaca catcctccag cacagcctac 240atgcagctca
gcaggctgac atctgaggac tctgctgtct atttctgtgc aagacagact
300acggctacct ggtttgctta ctggggccaa gggaccacgg tcaccgtctc
ctcagatgga 360ggcggttcag gcggaggtgg ctctggcggt ggcggatcgg
acatcgagct cactcagtct 420ccaaccaccc tgtctgtgac tccaggagat
agagtctctc tttcctgcag ggccagccag 480agtattagcg actacttaca
ctggtaccaa caaaaatcac atgagtctcc aaggcttctc 540atcaaatatg
cttcccaatc catctctggg atcccctcca ggttcagtgg cagtggatca
600gggtcagatt tcactctcag tatcaacagt gtggaacctg aagatgttgg
agtgtattac 660tgtcaaaatg gtcacagctt tccgctcacg ttcggtgctg
ggaccaagct ggagctgaaa 720caggcggccg c 7312243PRTMus musculus 2Gln
Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10
15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30Asp Ile Asn Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu Trp
Ile 35 40 45Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu
Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Thr Asp Thr Ser Ser Ser
Thr Ala Tyr65 70 75 80Met Gln Leu Ser Arg Leu Thr Ser Glu Asp Ser
Ala Val Tyr Phe Cys 85 90 95Ala Arg Gln Thr Thr Ala Thr Trp Phe Ala
Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Asp Gly
Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Asp
Ile Glu Leu Thr Gln Ser Pro Thr Thr Leu 130 135 140Ser Val Thr Pro
Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln145 150 155 160Ser
Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser 165 170
175Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro
180 185 190Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu
Ser Ile 195 200 205Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr
Cys Gln Asn Gly 210 215 220His Ser Phe Pro Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys225 230 235 240Gln Ala Ala3243PRTArtificial
SequenceMutated 8H9 scFv with decreased normal tissue adherence
3Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Glu Pro Gly Ala1 5
10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30Asp Ile Asn Trp Val Arg Gln Arg Pro Glu Gln Gly Leu Glu
Trp Ile 35 40 45Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Gln Tyr Asn
Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Thr Asp Thr Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Arg Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Gln Thr Thr Ala Thr Trp Phe
Ala Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Asp
Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser
Asp Ile Glu Leu Thr Gln Ser Pro Thr Thr Leu 130 135 140Ser Val Thr
Pro Gly Asp Gln Val Ser Leu Ser Cys Arg Ala Ser Gln145 150 155
160Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser
165 170 175Pro Gln Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly
Ile Pro 180 185 190Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe
Thr Leu Ser Ile 195 200 205Asn Ser Val Glu Pro Glu Asp Val Gly Val
Tyr Tyr Cys Gln Asn Gly 210 215 220His Ser Phe Pro Leu Thr Phe Gly
Ala Gly Thr Glu Leu Glu Leu Glu225 230 235 240Gln Ala
Ala422DNAArtificial Sequence[32P]r Probe 4tactctcagc agaacaccta tg
22521DNAArtificial SequencePrimer ESBP1 5cgactagtta tgatcagagc a
21623DNAArtificial SequencePrimer ESBP2 6ccgttgctct gtattcttac tga
23718DNAArtificial SequencePrimer EWS 696 7agcagctatg gacagcag
18820DNAArtificial SequencePrimer FLI 1 1041 8ttgaggccag aattcatgtt
20925DNAArtificial SequencePrimer G6PD1 9ccggatcgac cactacctgg
gcaag 251026DNAArtificial SequencePrimer G6PD2 10gttccccacg
tactggccca ggacca 261124DNAArtificial SequenceLightcycler
Hybridization Probe EWSHP1 11tatagccaac agagcagcag ctac
241218DNAArtificial SequenceLightcycler Hybridization Probe EWSHP2
12ggcagcagaa cccttctt 181328DNAArtificial SequenceLightcycler
Hybridization Probe G6PDHP1 13gttccagatg gggccgaaga tcctgttg
281428DNAArtificial SequenceLightcycler Hybridization Probe G6PDHP2
14caaatctcag caccatgagg ttctgcac 281532DNAArtificial
SequenceSynthetic sequence 15ttattacgag ttacatggcc ttaccagtga cc
321632DNAArtificial SequenceSynthetic sequence 16ttattacgag
taacatggcc ttaccagtga cc 321731DNAArtificial SequenceSynthetic
sequence 17cttggtccga gtgtcaggag cgataggctg c 311831DNAArtificial
SequenceSynthetic 18cttggttcga gtgtcaggag cgataggctg c
311930DNAArtificial SequenceSynthetic sequence 19ttattacgaa
tgattgccca ggtcaaactg 302030DNAArtificial SequenceSynthetic
sequence 20ttattacgaa cgattgccca ggtcaaactg 302128DNAArtificial
SequenceSynthetic sequence 21cttggtgggc cgcctgtttc agctccag
282228DNAArtificial SequenceSynthetic sequence 22cttggtcggc
cgcctgtttc agctccag 282346DNAArtificial SequenceSynthetic sequence
23cggacttagc agcctatcgc tcctggcacc gagaagagtg aagttc
462446DNAArtificial SequenceSynthetic sequence 24cggacttagc
agcctatcgc tcctggcatc gagaagagtg aagttc 462546DNAArtificial
SequenceSynthetic sequence 25ccgacttagc agcctatcgc tcctggcacc
gagaagagtg aagttc 462646DNAArtificial SequenceSynthetic sequence
26ccgacttagc agcctatcgc tcctggcatc gagaagagtg aagttc
462729DNAArtificial SequenceSynthetic sequence 27cttggtaatc
ttcagcgagg gggcagggc 292829DNAArtificial SequenceSynthetic sequence
28cttggtgatc ttcagcgagg gggcagggc 29295PRTMus musculus 29Asn Tyr
Asp Ile Asn1 53011PRTMus musculus 30Trp Ile Phe Pro Gly Asp Gly Ser
Thr Gln Tyr1 5 10319PRTMus musculus 31Gln Thr Thr Ala Thr Trp Phe
Ala Tyr1 53211PRTMus musculus 32Arg Ala Ser Gln Ser Ile Ser Asp Tyr
Leu His1 5 10337PRTMus musculus 33Tyr Ala Ser Gln Ser Ile Ser1
5349PRTMus musculus 34Gln Asn Gly His Ser Phe Pro Leu Thr1
535243PRTMus musculus 35Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu
Val Lys Pro Gly 5 10 15Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr 20 25 30Asn Tyr Asp Ile Asn Trp Val Arg Gln Arg Pro
Glu Gln Gly Leu 35 40 45Glu Trp Ile Gly Trp Ile Phe Pro Gly Asp Gly
Ser Thr Gln Tyr 50 55 60Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr
Thr Asp Thr Ser 65 70 75Ser Ser Thr Ala Tyr Met Gln Leu Ser Arg Leu
Thr Ser Glu Asp 80 85 90Ser Ala Val Tyr Phe Cys Ala Arg Gln Thr Thr
Ala Thr Trp Phe 95 100 105Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Gly Gly 110 115 120Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile 125 130 135Glu Leu Thr Gln Ser Pro Thr Thr
Leu Ser Val Thr Pro Gly Asp 140 145 150Arg Val Ser Leu Ser Cys Arg
Ala Ser Gln Ser Ile Ser Asp Tyr 155 160 165Leu His Trp Tyr Gln Gln
Lys Ser His Glu Ser Pro Arg Leu Leu 170 175 180Ile Lys Tyr Ala Ser
Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe 185 190 195Ser Gly Ser Gly
Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser 200 205 210Val Glu Pro
Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His 215 220 225Ser Phe
Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 230 235 240Gln
Ala Ala 24336731DNAMus musculus 36caggtcaaac tgcagcagtc tggggctgaa
ctggtaaagc ctggggcttc agtgaaattg 60tcctgcaagg cttctggcta caccttcaca
aactatgata taaactgggt gaggcagagg 120cctgaacagg gacttgagtg
gattggatgg atttttcctg gagatggtag tactcaatac 180aatgagaagt
tcaagggcaa ggccacactg actacagaca catcctccag cacagcctac
240atgcagctca gcaggctgac atctgaggac tctgctgtct atttctgtgc
aagacagact 300acggctacct ggtttgctta ctggggccaa gggaccacgg
tcaccgtctc ctcaggtgga 360ggcggttcag gcggaggtgg ctctggcggt
ggcggatcgg acatcgagct cactcagtct 420ccaaccaccc tgtctgtgac
tccaggagat agagtctctc tttcctgcag ggccagccag 480agtattagcg
actacttaca ctggtaccaa caaaaatcac atgagtctcc aaggcttctc
540atcaaatatg cttcccaatc catctctggg atcccctcca ggttcagtgg
cagtggatca 600gggtcagatt tcactctcag tatcaacagt gtggaacctg
aagatgttgg agtgtattac 660tgtcaaaatg gtcacagctt tccgctcacg
ttcggtgctg ggaccaagct ggagctgaaa 720caggcggccg c 73137731DNAMus
musculus 37gtccagtttg acgtcgtcag accccgactt gaccatttcg gaccccgaag
tcactttaac 60aggacgttcc gaagaccgat gtggaagtgt ttgatactat atttgaccca
ctccgtctcc 120ggacttgtcc ctgaactcac ctaacctacc taaaaaggac
ctctaccatc atgagttatg 180ttactcttca agttcccgtt ccggtgtgac
tgatgtctgt gtaggaggtc gtgtcggatg 240tacgtcgagt cgtccgactg
tagactcctg agacgacaga taaagacacg ttctgtctga 300tgccgatgga
ccaaacgaat gaccccggtt ccctggtgcc agtggcagag gagtccacct
360ccgccaagtc cgcctccacc gagaccgcca ccgcctagcc tgtagctcga
gtgagtcaga 420ggttggtggg acagacactg aggtcctcta tctcagagag
aaaggacgtc ccggtcggtc 480tcataatcgc tgatgaatgt gaccatggtt
gtttttagtg tactcagagg ttccgaagag 540tagtttatac gaagggttag
gtagagaccc taggggaggt ccaagtcacc gtcacctagt 600cccagtctaa
agtgagagtc atagttgtca caccttggac ttctacaacc tcacataatg
660acagttttac cagtgtcgaa aggcgagtgc aagccacgac cctggttcga
cctcgacttt 720gtccgccggc g 73138731DNAMus musculus 38caggtcaaac
tgcagcagtc tggggctgaa ctggtaaagc ctggggcttc agtgaaattg 60tcctgcaagg
cttctggcta caccttcaca aactatgata taaactgggt gaggcagagg
120cctgaacagg gacttgagtg gattggatgg atttttcctg gagatggtag
tactcaatac 180aatgagaagt tcaagggcaa ggccacactg actacagaca
catcctccag cacagcctac 240atgcagctca gcaggctgac atctgaggac
tctgctgtct atttctgtgc aagacagact 300acggctacct ggtttgctta
ctggggccaa gggaccacgg tcaccgtctc ctcagatgga 360ggcggttcag
gcggaggtgg ctctggcggt ggcggatcgg acatcgagct cactcagtct
420ccaaccaccc tgtctgtgac tccaggagat agagtctctc tttcctgcag
ggccagccag 480agtattagcg actacttaca ctggtaccaa caaaaatcac
atgagtctcc aaggcttctc 540atcaaatatg cttcccaatc catctctggg
atcccctcca ggttcagtgg cagtggatca 600gggtcagatt tcactctcag
tatcaacagt gtggaacctg aagatgttgg agtgtattac 660tgtcaaaatg
gtcacagctt tccgctcacg ttcggtgctg ggaccaagct ggagctgaaa
720caggcggccg c 73139243PRTMus musculus 39Gln Val Lys Leu Gln Gln
Ser Gly Ala Glu Leu Val Lys Pro Gly 5 10 15Ala Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 30Asn Tyr Asp Ile Asn Trp
Val Arg Gln Arg Pro Glu Gln Gly Leu 35 40 45Glu Trp Ile Gly Trp Ile
Phe Pro Gly Asp Gly Ser Thr Gln Tyr 50 55 60Asn Glu Lys Phe Lys Gly
Lys Ala Thr Leu Thr Thr Asp Thr Ser 65 70 75Ser Ser Thr Ala Tyr Met
Gln Leu Ser Arg Leu Thr Ser Glu Asp 80 85 90Ser Ala Val Tyr Phe Cys
Ala Arg Gln Thr Thr Ala Thr Trp Phe 95 100 105Ala Tyr Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Asp Gly 110 115 120Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 125 130 135Glu Leu Thr
Gln Ser Pro Thr Thr Leu Ser Val Thr Pro Gly Asp 140 145 150Arg Val
Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr 155 160 165Leu
His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu 170 175
180Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe 185
190 195Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser
200 205 210Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly
His 215 220 225Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 230 235 240Gln Ala Ala 24340243PRTArtificial
SequenceMutated 8H9 scFv with decreased normal tissue adherence
40Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Glu Pro Gly 5 10
15Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25
30Asn Tyr Asp Ile Asn Trp Val Arg Gln Arg Pro Glu Gln Gly Leu 35 40
45Glu Trp Ile Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Gln Tyr 50 55
60Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Thr Asp Thr Ser 65 70
75Ser Ser Thr Ala Tyr Met Gln Leu Ser Arg Leu Thr Ser Glu Asp 80 85
90Ser Ala Val Tyr Phe Cys Ala Arg Gln Thr Thr Ala Thr Trp Phe 95
100 105Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Asp Gly
110 115 120Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
Ile 125 130 135Glu Leu Thr Gln Ser Pro Thr Thr Leu Ser Val Thr Pro
Gly Asp 140 145 150Gln Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile
Ser Asp Tyr 155 160 165Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser
Pro Gln Leu Leu 170 175 180Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly
Ile Pro Ser Arg Phe 185 190 195Ser Gly Ser Gly Ser Gly Ser Asp Phe
Thr Leu Ser Ile Asn Ser 200 205 210Val Glu Pro Glu Asp Val Gly Val
Tyr Tyr Cys Gln Asn Gly His 215 220 225Ser Phe Pro Leu Thr Phe Gly
Ala Gly Thr Glu Leu Glu Leu Glu 230 235 240Gln Ala Ala 243
* * * * *